Compositions of polymeric myrcene

ABSTRACT

Compositions and formulations comprising polymeric myrcene. More particularly, the invention relates to compositions comprising an isolated fraction of polymeric myrcene in a hydrophobic carrier and formulations which maintain the biological activity of the active polymer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 14/226,293 filedMar. 26, 2014, which is a continuation of U.S. patent application Ser.No. 13/254,380, filed Sep. 1, 2011, now U.S. Pat. No. 8,722,105, whichis a 35 U.S.C. §371 National Phase Entry Application fromPCT/IL2010/000184, filed Mar. 4, 2010, and designating the UnitedStates, which claims the benefit of U.S. Patent Application No.61/157,216 filed Mar. 4, 2009, all of which are incorporated herein intheir entireties.

FIELD OF THE INVENTION

The invention relates to compositions isolated from mastic gum, andtheir therapeutic use. More particularly, the invention relates tocompositions comprising an isolated fraction of polymeric myrcene andformulations which maintain the biological activity of the activepolymer.

BACKGROUND OF THE INVENTION

The pursuit of new drug entities derived from plants and plant productsfor various therapeutic applications has its origins in antiquity andcontinues to the present. One such source is mastic, also known as gummastic or mastic gum, which is a tree resin obtained as an exudate fromPistacia lentiscus L., a member of the family Anacardiaceae. Mastic wasused in the ancient Mediterranean world for gastrointestinal disorderssuch as gastralgia, dyspepsia and peptic ulcer. Oral administration ofmastic to human patients with duodenal ulcer and to experimental ratswith induced gastric and duodenal ulcers has been disclosed to havetherapeutic effects (Al-Habbal et al (1984) Clin Exp Pharmacop Physio11(5):541-4; Said et al (1986) J Ethnopharmacol 15(3):271-8). While ithas been disclosed that mastic has in vitro bactericidal effects againstHelicobacter pylori, the etiologic agent causing peptide ulcer disease(Marone et al (2001) J Chemother 13:611-614), other reports disclosethat mastic does not exert anti-bacterial activity upon administrationto H. pylori positive human patients (Bebb et al (2003) J AntimicrobChemother 52:522-23) or to experimentally infected mice (Loughlin et al(2003) J Antimicrob Chemother 51:367-371).

Greek Patent No. GR 1,003,541 discloses antimicrobial and antifungalaction of the chios mastic oil extracted from the leaves, branches andfruit of Pistacia lentiscus var Chia.

Greek Patent No. GR 1,003,868 discloses use of a product derived fromPistacia lentiscus var. Chia as an antioxidant, as a wound healinginductor and as a cytostatic agent.

U.S. Patent Application Publication No 2005/0238740 discloses thatcertain fractions extracted from mastic resin exhibit anti-microbial andanti-cell proliferative activities. According to the disclosure, a firstextract (termed “total fraction” or “whole extract”) contains all thecompounds of the mastic gum except the polymer resin; a second extractis an acid fraction containing all the triterpenic acids of the totalfraction, and a third extract is a neutral fraction containing all theother terpenes of the total fraction. Additionally disclosed is anessential oil obtained by distillation which contains monoterpenes,inter alia, β-myrcene. The application discloses pharmaceuticalformulations comprising any of the aforementioned total, acid or neutralfractions optionally combined with the essential oil, or syntheticequivalents thereof, or comprising isolated component products orsynthetic equivalents thereof, as well as the use thereof in methods fortreating microbial infections including H. pylori, Propionibacterium,Staphylococcus, Pseudomonas, and cell hyperproliferation.

Paraschos et al (2007), authored by some of the inventors of theaforementioned patent application, disclose preparation of a totalmastic extract without polymer (TMEWP), prepared by polar solventextraction of crude mastic and removal of the insoluble polymerpoly-β-myrcene therefrom, and acidic and neutral fractions separatedfrom TMEWP (Paraschos et al (2007) Antimicrob Agents Chemother51(2):551-559). According to the disclosure, administration of TMEWP toH. pylori infected mice over a period of 3 months resulted in a 30-foldreduction of bacterial colonization, largely attributable to aparticular compound purified from the acid fraction. The authorsindicate that TMEWP was prepared since the high percentage ofpoly-β-myrcene in crude mastic preparations, as used in previousstudies, was speculated to hinder potential in vivo activity during oraladministration. The authors further disclose that removal of thepoly-β-myrcene can produce an enhanced therapeutic moiety with anti-H.pylori activity.

EP Patent Application No. 1520585 discloses use of a product obtainedfrom a plant of the genus Pistacia for the manufacture of a medicamentfor treating or preventing cancer. According to the disclosure,essential oils distilled from leaves, twigs, fruits, nuts and flowers ofdifferent Pistacia species contain a large number of monomeric terpenecompounds in varying proportions inter alia β-myrcene. The applicationfurther discloses that the oils have activity against certain tumorcells lines such as colon and ovary adenocarcinomas, and that bornylacetate was the only single component found to have anti-canceractivity.

International Patent Application Publication No. WO 2005/112967discloses the purification from mastic of an anti-cancer compound havinganti-proliferative effects, which is found in a soluble fractionobtained by suspending mastic in a solvent selected from a non-acidic,aliphatic hydrocarbon, an aqueous solution containing at least 25% of awater-soluble, non-acidic, aliphatic hydrocarbon, or a combinationthereof, and removing the insoluble fraction. The application furtherdiscloses a method for treating cancer cells comprising administering apharmaceutically effective amount of a fraction of mastic gum resin thatinhibits growth of cancer cells. According to the disclosure, theanti-cancer compound is effective against various types of cancer cells,including human colon cancer cells.

Van der Berg et al (1998) disclose isolation and purification of thepolymer fraction of mastic using extraction and size exclusionchromatography (Van der Berg et al (1998) Tetrahedron Lett 3:2645-2648).According to the disclosure, the polymer has a broad molecular weightdistribution i.e. 20,000 to 100,000 Da, is formed from monomer units of136 Da, and has the structure of 1,4-poly-β-myrcene, with cis- andtrans-configurations at a ratio of 3:1. The authors assert that theirdisclosure is the first report of a naturally occurring polymer of amonoterpene.

Barra et al (2007) disclose extraction and gas chromatographic analysisof essential oil from P. lentiscus L. (Barra et al (2007) J Agric FoodChem 55(17):7093-7098). According to the disclosure, a total of 45compounds were identified, including β-myrcene as one of the majorcompounds.

Marner et al (1991) disclose identification of various triterpenoidsfrom gum mastic of P. lentiscus (Marner et al (1991) Phytochemistry, 30,3709-3712).

U.S. Pat. No. 5,506,406 discloses mastic oil produced by dissolvingmastic in an oil or fat, and filled in a soft capsule which optionallyfurther contains an amphipathic substance such as chitin or chitosan.According to the disclosure, the capsule is effective for eliminatingand inhibiting H. pylori, and for reducing the smell of feces. U.S. Pat.No. 5,637,290 discloses an oral hygiene product comprising thecombination of a toothpaste and an ingredient selected from naturalmastic, extracted mastic oil and synthetic mastic oil agents. Alsodisclosed is use of mastic for incorporation into suntan lotion, hairproducts and cosmetics.

U.S. Pat. No. 6,623,728 discloses cosmetic skin care emulsioncompositions comprising from about 0.001 wt % to about 10 wt %solubilized gum mastic; a volatile water miscible solvent; and acosmetically acceptable vehicle. According to the disclosure, theemulsion is preferably an oil-in-water emulsion, and preferred solventsinclude ethanol, methanol propanol, isopropyl alcohol and mixturesthereof. According to the disclosure, the same types of solvents areused to obtain the solubilized gum mastic.

U.S. Pat. No. 6,811,769 discloses an oral composition comprising an oilextract of mastic, such as that prepared with olive oil or palm oil; andan antiphlogistic, such as glycyrrhizic acid. According to thedisclosure, the composition has antibacterial action against periodontaldisease-related bacteria and against tooth decay-related bacteria.

U.S. Pat. No. 7,294,651 discloses use of isoprenoid compounds, interalia, terpene compounds for inhibiting the production of exoproteins ofGram positive bacteria, such as Toxic Shock Syndrome Toxin-1 produced byS. aureus. According to the disclosure, suitable terpenes may be cyclicor acyclic, saturated or unsaturated, and also include polyterpenes.Also disclosed is the use of such compounds for preparing compositionswhich may be incorporated into aqueous solutions, such as vaginalcleaning formulations.

U.S. Pat. No. 4,564,718 discloses preparation of functionally terminatedpolymers, referred to as “liquid rubbers” having glass transitiontemperatures substantially less than room temperature, by polymerizationof a terpene or oxygen derivative thereof having a double bond orconjugated double bond available for polymerization, with an initiatorwhich provides the desired functional termination. According to thedisclosure, the polymers preferably have a molecular weight of 500 to20,000, and preferred acyclic monoterpenes for preparation thereof areinter alia β-myrcene. The patent discloses preparation of polymericmyrcene of molecular weight of about 2000 and of about 4000. The patentfurther discloses that the polymers of the invention may be furtherreacted with other reagents to provide elastomers, sealants oradhesives, or they may be used as rubber toughening agents. Furtherdisclosed is preparation of hydroxy-terminated polymyrcene from myrcene,and use thereof to prepare a polyurethane elastomer.

Newmark et al J. Polymer Sci. 26, 71-77 (1988) discloses synthesis ofpolymyrcene having an observed molecular weight of 87,000 and acalculated molecular weight of 46,000.

U.S. Pat. No. 4,374,957 discloses a tacky thermoplastic elastomericlinear triblock polymer corresponding to the formula A-B-A, wherein A isa nonelastic linear homopolymer block of a monovinyl aromatichydrocarbon having an average molecular weight between 10,000 and 60,000and a glass transition temperature above 70° C., and wherein B is anelastomeric homopolymeric block of myrcene having an average molecularweight between 50,000 and 200,000 and a glass transition temperaturebelow about −40° C.

U.S. Pat. No. 5,759,569 discloses biodegradable compostable articlesthat at least partially comprise certain trans-polymers, wherein thepolymers have a weight average molecular weight of at least about 20,000and are made by polymerizing a monomer component that comprises: (1)from about 70 to 100 mole % 1,3-dienes inter alia β-myrcene; and (2) upto about 30 mole % other compatible co monomers. According to thedisclosure, the articles include inter alia packaging materials;disposable absorbent articles (e.g., diapers, sanitary napkins); garmentarticles such as protective clothing, surgical drapes, surgical gowns,surgical sheets; woven, knitted and non-woven fabrics; surgical sponges,tampon applicators, disposable syringes and containers.

U.S. Pat. Nos. 7,232,872 and 7,214,750 disclose a polymerization processcomprising contacting one or more monomer(s) inter alia myrcene, one ormore Lewis acid(s), one or more initiator(s), and a diluent comprisingone or more hydrofluorocarbon(s) in a reactor.

U.S. Patent Application Publication No 2007/0179260 and U.S. Pat. No.7,417,103 disclose 3,4-isoprene-based polymers with high regioregularityand a method for producing same. According to these disclosures, thenumber average molecular weight of the polymer is 5000 to 6,000,000, andthe polymer may also include units of 1,4-isoprenes such as myrcene.According to the disclosure, the polymer is suitable for use as aplastic material due to its mechanical and thermal durability.

The prior art does not contain any teaching or suggestion of the use ofan isolated fraction of polymeric myrcene, whether that derived frommastic, or that chemically synthesized, as an active ingredient in apharmaceutical composition or in a therapeutic application. The priorart does not teach or suggest use of an isolated fraction of polymericmyrcene in a composition for treating neurological conditions or skindisorders.

SUMMARY OF THE INVENTION

The present invention provides pharmaceutical compositions comprisingpolymeric forms of the monoterpene compound known as myrcene, whichexhibit a variety of beneficial biological activities which may beexploited for therapeutic applications. More specifically, compositionscomprising isolated fractions of polymeric myrcene, including thatchemically synthesized and that derived from plant sources such asmastic gum, are now disclosed to have activity in neuroprotection,tissue regeneration and wound and tissue repair.

The compositions disclosed herein may be prepared by solvent extractionof certain plant material, such as mastic gum, in so as to obtainisolated fractions which are soluble in both polar and non-polarsolvents, and are depleted of various monomeric terpene compounds whichinterfere with the desired biological activity.

The teachings of the present invention have been exemplified with masticgum extracts prepared by a two-step extraction procedure, so as toobtain a fraction that is soluble in both a polar solvent and anon-polar solvent, and wherein material from the mastic gum that issoluble in the polar solvent but remains insoluble in the non-polarsolvent is eliminated. The present invention has also been exemplifiedboth with polymeric myrcene isolated from a natural source i.e. treeresin mastic, and with chemically synthesized polymeric myrcene havingthe molecular weight in the same range and chemical structure as that ofthe corresponding polymer isolated from mastic.

Moreover, the teachings of the present invention are particularlysurprising and unexpected over teachings which disclose the use ofmastic gum extract fractions from which polymeric myrcene has beenremoved. Furthermore, the prior art asserts that polymeric fractionsderived from mastic are not therapeutically useful, and that thepresence of polymeric myrcene in therapeutic compositions actuallyinhibits the beneficial biological activities and bioavailability of theactive compounds. The prior art teaches that the active compounds inmastic gum correspond to various low molecular weight terpene-typemolecules, inter alia monomeric myrcene. However, the inventors of thepresent invention have surprisingly found, and contrary to the teachingsof the prior art, that monomeric myrcene, small oligomeric forms ofmyrcene, and certain other low molecular weight terpenes interfere withand block the activity of polymeric myrcene in inducing celldifferentiation.

Without wishing to be bound by any particular theory or mechanism ofaction, the activity of polymeric myrcene for induction of neuronal celldifferentiation, as disclosed herein, renders the present inventionuseful for reformation of inter-neuronal junctions and overcomingdefective inter-neuronal communication in brain and neural tissueaffected by pathologies associated with inadequate synaptic formation.This pathology underlies many nervous system pathologies, including forexample Alzheimer's disease. The invention is further useful forpromoting wound healing and rejuvenation of a large number of cells andtissues.

As used herein “polymeric myrcene” encompasses polymeric forms ofmyrcene having a degree of polymerization of at least 6. Polymericmyrcene includes without limitation, polymeric β-myrcene(poly-β-myrcene), polymeric α-myrcene (poly-α-myrcene), homopolymersthereof and heteropolymers (also known as copolymers) which containmyrcene subunits. Also included are geometric isomers, optical isomersand diastereoisomers of polymeric myrcene compounds.

It is to be understood explicitly that the scope of the presentinvention does not include myrcene in its monomeric form, such asβ-myrcene and α-myrcene, as active ingredients of the fractions andcompositions disclosed herein.

As used herein, β-myrcene refers to 7-methyl-3-methylene-1,6-octadieneand α-myrcene refers to the structural isomer2-methyl-6-methylene-1,7-octadiene.

It is to be further understood that the biological activity of thefractions and compositions disclosed herein is inhibited by the presenceof certain monomeric and small oligomeric forms of various terpenes.

According to a first aspect, the present invention provides acomposition comprising an effective amount of an isolated fraction ofmastic gum, wherein the fraction is characterized in that it is solublein at least one polar organic solvent and in at least one non-polarorganic solvent, and wherein said fraction is substantially devoid ofcompounds which are soluble in said polar organic solvent but insolublein said non-polar organic solvent.

In a particular embodiment, the composition is obtained by a processcomprising the steps of:

-   -   (a) treating mastic gum with a polar organic solvent;    -   (b) isolating a fraction soluble in said polar organic solvent;    -   (c) optionally removing said polar organic solvent;    -   (d) treating the soluble fraction obtained in step (b) or (c)        with a non-polar organic solvent, (e) isolating a fraction        soluble in said nonpolar organic solvent; and    -   (f) optionally removing said nonpolar organic solvent;    -   wherein steps (d) to (f) may precede steps (a) to (c).

In particular embodiments, steps (a) to (c) are carried out prior tosteps (d) to (f); or steps (d) to (f) are carried out prior to steps (a)to (c). In particular embodiments, (a) to (c) and/or steps (d) to (f)are repeated for a multiplicity of cycles.

In a particular embodiment, either or both of steps (c) and (f) compriseremoving the solvent by a means selected from the group consisting ofrotary evaporation, application of high vacuum and a combinationthereof. In a particular embodiment, the process further comprises thestep of size fractionating the fraction obtained by said process.

Polar organic solvents suitable for use in the invention may be selectedfrom an alcohol, an ether, an ester, an amide, an aldehyde, a ketone, anitrile, and combinations thereof.

Specific examples of suitable polar organic solvents include methanol,ethanol, propanol, isopropanol, 1-butanol, 2-butanol, sec-butanol,t-butanol, 1-pentanol, 2-pentanol, 3-pentanol, neopentanol,3-methyl-1-butanol, 2-methyl-1-butanol, 3-methyl-2-butanol,2-methyl-2-butanol, ethyleneglycol, ethyleneglycol monomethyl ether,diethyl ether, methylethyl ether, ethylpropyl ether, methylpropyl ether,1,2-dimethoxyethane, tetrahydrofuran, dihydrofuran, furan, pyran,dihydropyran, tetrahydropyran, methyl acetate, ethyl acetate, propylacetate, acetaldehyde, methylformate, ethylformate, ethyl propionate,methyl propionate, dichloromethane, chloroform, dimethylformamide,acetamide, dimethylacetamide, N-methylpyrrolidone, acetone, ethylmethylketone, diethyl ketone, acetonitrile, propionitrile, and combinationsthereof.

In particular embodiments, the polar organic solvent is ethanol.

Non-polar organic solvents suitable for use in the invention may beselected from acyclic or cyclic, saturated or unsaturated aliphatichydrocarbons and aromatic hydrocarbons, each of which is optionallysubstituted by one or more halogens, and combinations thereof. Inparticular embodiments, the non-polar organic solvent is selected fromC5-C10 alkanes, C5-C10 cycloalkanes, C6-C14 aromatic hydrocarbons andC7-C14 perfluoroalkanes, and combinations thereof.

In particular embodiments, the non-polar organic solvent is selectedfrom pentanes, hexanes, heptanes, octanes, nonanes, decanes,cyclopentane, cyclohexane, cycloheptane, benzene, toluene, xylene, andisomers and mixtures thereof.

In particular embodiments, the C5-C10 alkane is selected from the groupconsisting of pentane, hexane, heptane, octane, nonane, decane,cyclohexane, and isomers and mixtures thereof.

In particular embodiments, the non-polar organic solvent is hexane.

In a particular embodiment, the polar organic solvent comprises ethanoland the non-polar organic solvent comprises hexane.

In a particular embodiment, the composition is substantially devoid ofterpene compounds which are soluble in said polar organic solvent andinsoluble in said non-polar organic solvent. In a particular embodiment,the terpene compounds are monomeric terpene compounds. In a particularembodiment, the terpene compounds are selected from β-myrcene,α-myrcene, cis-α-ocimene, dihydromyrcene, limonene, α-pinene, β-pineneand combinations thereof.

In a particular embodiment, the composition comprises from about 0.01 toabout 25% (w/w) of the isolated fraction of mastic gum, based on thetotal weight of the composition. In a particular embodiment, thecomposition comprises from about 0.01 to about 12% (w/w) of the isolatedfraction of mastic gum, based on the total weight of the composition.

In a particular embodiment, the isolated fraction of gum masticcomprises polymeric myrcene.

In another aspect the invention provides a pharmaceutical compositioncomprising a therapeutically effective amount of an isolated fraction ofpolymeric myrcene, and a pharmaceutically acceptable carrier.

In a particular embodiment, the composition comprises from about 0.01 toabout 12% (w/w) polymeric myrcene, based on the total weight of thecomposition.

In particular embodiments, the polymeric myrcene is selected from thegroup consisting of polymeric β-myrcene (poly-β-myrcene), polymericα-myrcene (poly-α-myrcene), myrcene copolymers and combinations thereof.In particular embodiments, the poly-β-myrcene is selected from the groupconsisting of 1,4-poly-β-myrcene, 3,4-poly-β-myrcene, 1,2-poly-β-myrceneand combinations thereof. In particular embodiments, the polymericmyrcene comprises a myrcene isomer selected from the group consisting ofa cis isomer, a trans isomer and combinations thereof. In particularembodiments, the 1,4-poly-β-myrcene is selected from the groupconsisting of cis-1,4-poly-β-myrcene, trans-1,4-poly-β-myrcene andcombinations thereof. In particular embodiments, the polymeric myrcenecomprises cis-1,4-poly-β-myrcene. In particular embodiments, thepolymeric myrcene has a cyclic conformation. In particular embodiments,the polymeric myrcene has a branched conformation.

In a particular embodiment, the polymeric myrcene has a degree ofpolymerization in the range of at least about 6 to about 1800. In aparticular embodiment, the degree of polymerization is at least about10. In a particular embodiment, the degree of polymerization is at leastabout 15. In a particular embodiment, the degree of polymerization is atleast about 25. In a particular embodiment, the degree of polymerizationis at least about 35. In a particular embodiment, the degree ofpolymerization is in the range of about 6 to about 30. In a particularembodiment, the degree of polymerization is in the range of about 30 toabout 500, for example, in the range of about 33 to about 150.

Each possibility represents a separate embodiment of the invention.

In a particular embodiment, the polymeric myrcene has a number averagemolecular weight of at least about 800. In a particular embodiment, thenumber average molecular weight is at least about 1,000. In a particularembodiment, the number average molecular weight is at least about 2000.In a particular embodiment, the number average molecular weight is atleast about 3000. In a particular embodiment, the number averagemolecular weight is at least about 5000. In a particular embodiment, thepolymeric myrcene has a number average molecular weight in the rangefrom at least about 800 to about 100,000. In particular embodiments, thenumber average molecular weight is in a range selected from the groupconsisting of: at least about 800 to about 80,000; at least about 800 toabout 50,000; at least about 800 to about 20,000; at least about 800 toabout 10,000; at least about 800 to about 5000; at least about 1,000 toat least about 50,000; at least about 1,000 to about 10,000; at leastabout 1,000 to about 5000; about 5000 to about 10,000; about 10,000 toabout 15,000; about 5000 to about 20,000; about 15,000 to about 30,000;about 25,000 to about 40,000; about 35,000 to about 50,000; about 45,000to about 60,000; about 55,000 to about 70,000; about 65,000 to about80,000; about 75,000 to about 90,000; about 85,000 to about 100,000; orany combinations and sub-ranges thereof. In a particular embodiment, thepolymeric myrcene has a number average molecular weight in the rangefrom about 5,000 to about 20,000.

Each possibility represents a separate embodiment of the invention.

It is to be understood that the composition may comprise differentmolecular weight fractions of polymeric myrcene, for example in therange from at least about 800 to about 100,000, or various combinationsthereof. In a particular embodiment, the polymeric myrcene has apolydispersity index less than 5.

In a particular embodiment, the polymeric myrcene is the product of achemical synthesis. In a particular embodiment, the chemical synthesiscomprises use of monomeric myrcene as a substrate. In a particularembodiment, the substrate is β-myrcene. In a particular embodiment, theβ-myrcene substrate is derived from a plant.

In a particular embodiment, the product of the chemical synthesiscomprises cis-1,4-poly-β-myrcene. In a particular embodiment, thechemical synthesis comprises an anionic polymerization reaction. In aparticular embodiment, the chemical synthesis further comprisesdissolving the polymeric myrcene obtained therefrom in a hydrophobiccarrier, such as at least one vegetable oil.

In a particular embodiment, the isolated fraction of polymeric myrceneis derived from a natural source. Natural sources include plantsclassified in the family Anacardiaceae. In a particular embodiment, gummastic is from a plant classified in the family Anacardiaceae. Suitableplants include those classified in a genus selected from the groupconsisting of Pistacia, Pinus, Picea, Juniperus, Alsies, Larix,Antirrhinum, Boswellia, Citrus and Gynura. In a particular embodiment,the species of Pistacia is selected from the group consisting of P.lentiscus, P. atlantica, P. palestina, P. saportae, P. terebinthus, P.vera and P. integerrima. In a particular embodiment, the species ofPistacia is Pistacia lentiscus L. In a particular embodiment, thenatural source is a plant material selected from the group consisting ofresin, leaves, twigs, roots, flowers, seeds, buds, bark, nuts and roots.In a particular embodiment, the natural source is a plant classified ina genus selected from the group consisting of Ocimum, Laurus andLavendula.

In a particular embodiment, the isolated fraction of polymeric myrceneis obtained by a process comprising the steps of:

-   -   (a) contacting plant material with at least one polar organic        solvent;    -   (b) isolating a fraction which is soluble in the at least one        polar organic solvent    -   (c) optionally removing said polar organic solvent;    -   (d) treating the soluble fraction obtained in step (b) or (c)        with at least one non-polar organic solvent;    -   (e) isolating a fraction soluble in said nonpolar organic        solvent; and    -   (f) optionally removing said nonpolar organic solvent;

wherein steps (d) to (f) may precede steps (a) to (c), and wherein steps(a) to (c) and steps (d) to (f) are each independently carried out for anumber of cycles; so as to obtain an isolated fraction of polymericmyrcene.

In particular embodiments, the isolated fraction of polymeric myrcenehas a degree of purity of at least about 80% (w/w). The isolatedfraction of polymeric myrcene may have a degree of purity of at leastabout 85% (w/w). In particular embodiments, the isolated fraction ofpolymeric myrcene has a degree of purity of at least about 90% (w/w), orat least about 93%, or at least about 95%, or at least about 97%, or atleast about 98% or at least about 99%.

In a particular embodiment, the isolated fraction of polymeric myrcenehas a degree of purity of at least 80%, and the polymeric myrcene has adegree of polymerization of at least 6.

In a particular embodiment, the isolated fraction of polymeric myrcenehas a degree of purity of at least 90%, and the polymeric myrcene has adegree of polymerization of at least 10.

In a particular embodiment, the isolated fraction of polymeric myrcenecomprises at least 90% (w/w) of cis-1,4-poly-β-myrcene. In a particularembodiment, the isolated fraction of polymeric myrcene comprises amixture of cis-1,4-poly-β-myrcene and trans-1,4-poly-β-myrcene, whereinthe mixture comprises at least 50% (w/w) of cis-1,4-poly-β-myrcene. In aparticular embodiment, the isolated fraction of polymeric myrcenecomprises at least 90% (w/w) of cis-1,4-poly-β-myrcene having a numberaverage molecular weight of at least 800, or at least 1,000, or at least5,000 or at least 10,000. In a particular embodiment, the isolatedfraction of polymeric myrcene comprises at least 80% (w/w) ofcis-1,4-poly-β-myrcene having a number average molecular weight in therange from about 800 to about 5,000. In a particular embodiment, theisolated fraction of polymeric myrcene comprises at least 90% (w/w) ofcis-1,4-poly-β-myrcene having a number average molecular weight in therange from about 1,000 to about 10,000. In a particular embodiment, theisolated fraction of polymeric myrcene comprises at least 90% (w/w) ofcis-1,4-poly-β-myrcene having a number average molecular weight in therange from about 5,000 to about 20,000.

In a particular embodiment, the isolated fraction of polymeric myrcenecomprises at least 90% (w/w) of cis-1,4-poly-β-myrcene having a numberaverage molecular weight in the range from about 10,000 to about 20,000.In a particular embodiment, the isolated fraction of polymeric myrcenecomprises at least 90% (w/w) of cis-1,4-poly-β-myrcene having a numberaverage molecular weight in the range from about 20,000 to about 30,000.In a particular embodiment, the isolated fraction of polymeric myrcenecomprises at least 90% (w/w) of cis-1,4-poly-β-myrcene having a numberaverage molecular weight in the range from about 30,000 to about 50,000In a particular embodiment, the isolated fraction of polymeric myrcenecomprises at least 90% (w/w) of cis-1,4-poly-β-myrcene having a numberaverage molecular weight in the range from about 50,000 to about 80,000.

In particular embodiments, the composition comprises less than about 10%(w/w), and more preferably, less than about 5% (w/w), of terpenecompounds which are soluble in a polar organic solvent and insoluble ina non-polar organic solvent. In particular embodiments, the compositionis substantially devoid of terpene compounds which are soluble in apolar organic solvent and insoluble in a non-polar organic solvent. Inparticular embodiments, the composition comprises less than about 10%(w/w), and more preferably, less than about 5% (w/w), of monomericterpene compounds. In a particular embodiment, the composition issubstantially devoid of myrcene monomers.

As referred to herein, terpene compounds include monomeric andoligomeric forms of terpene compounds, including those variouslyclassified as monoterpenes, diterpenes, sequiterpenes, triterpenes andtetraterpenes, including their acid, aldehyde and alcohol forms. In aparticular embodiment, the composition comprises less than about 10%(w/w), and more preferably, less than about 5% (w/w), of a terpenecompound selected from the group consisting of: β-myrcene, α-myrcene,cis-α-ocimene, dihydromyrcene, limonene, α-pinene, β-pinene andcombinations thereof.

In a particular embodiment, the isolated fraction of polymeric myrceneis derived from a plant and the composition is substantially devoid ofmyrcene monomers and myrcene oligomeric forms having a degree ofpolymerization less than about 6. In a particular embodiment, theisolated fraction of polymeric myrcene is derived from a plant and thecomposition is substantially devoid of terpene compounds which aresoluble in at least one polar organic solvent and insoluble in at leastone non-polar organic solvent.

In a particular embodiment, the isolated fraction of polymeric myrceneis the product of a chemical synthesis and the composition issubstantially devoid of myrcene monomers and myrcene oligomeric formshaving a degree of polymerization less than about 6. In a particularembodiment, the isolated fraction of polymeric myrcene is the product ofa chemical synthesis and the composition is substantially devoid ofterpene compounds which are soluble in a polar organic solvent andinsoluble in a non-polar solvent.

According to another aspect the present invention discloses apharmaceutical composition comprising a synthetic polymeric myrcenewherein the polymeric myrcene has a number average molecular weight inthe range of at least about 800 up to about 50,000, and wherein theisolated fraction of polymeric myrcene has a degree of purity of atleast 80%.

In a particular embodiment, the pharmaceutically acceptable carriercomprises a hydrophobic carrier. In a particular embodiment, thehydrophobic carrier comprises at least one oil. In a particularembodiment, the oil is selected from the group consisting of a mineraloil, a vegetable oil and combinations thereof. In a particularembodiment, the vegetable oil is selected from the group consisting ofalmond oil, canola oil, coconut oil, corn oil, cottonseed oil, grapeseed oil, olive oil peanut oil, saffron oil, sesame oil, soybean oil,and combinations thereof. In a particular embodiment, the mineral oil islight mineral oil. In a particular embodiment, the hydrophobic carriercomprises at least one wax. In a particular embodiment, the hydrophobiccarrier comprises a combination of at least one oil and at least onewax.

In various embodiments, a composition according to the invention is in aform suitable for administration by a route selected from the groupconsisting of oral, topical, parenteral and transdermal.

In particular embodiments, the composition is in a form suitable foradministration by injection. In various embodiments, the composition isa parenteral formulation for administration by a route selected from thegroup consisting of intravenous, intramuscular, subcutaneous,intradermal, intraperitoneal, intraarterial, intracerebral,intracerebroventricular, intraosseus and intrathecal. In variousembodiments, the composition is a topical formulation for administrationby a route selected from the group consisting of dermal, vaginal,rectal, inhalation, intranasal, ocular, auricular and buccal.

In particular embodiments, the composition is in a form suitable forcosmetic or dermatologic administration.

In particular embodiments, the pharmaceutical composition is in a formselected from the group consisting of a capsule, a tablet, a liposome, asuppository, a suspension, an ointment, a cream, a lotion, a solution,an emulsion, a film, a cement, a powder, a glue, an aerosol and a spray.In a particular embodiment, the capsule is selected from the groupconsisting of a hard gelatin capsule and a soft gelatin capsule. In aparticular embodiment, the emulsion is a nanoemulsion or amicroemulsion.

In particular embodiments, the formulation comprises at least one of aninclusion complex, a nanoemulsion, a microemulsion, a powder, a lipidraft, a lipid microparticle, a dendrimer and a liposome. In a particularembodiment, the inclusion complex comprises at least one cyclodextrin.In a particular embodiment, the at least one cyclodextrin compriseshydroxypropyl-β-cyclodextrin. In a particular embodiment, thenanoemulsion comprises droplets having average particle size of lessthan 800 nm. In a particular embodiment, the droplets have averageparticle size of less than 500 nm. In a particular embodiment, thedroplets have average particle size of less than 200 nm. In a particularembodiment, the powder is a spray dried powder. In a particularembodiment, the liposome comprises a multilamellar vesicle. In aparticular embodiment, the microemulsion comprises a non-ionicsurfactant. In a particular embodiment, the non-ionic surfactant isselected from the group consisting of a polyoxyl castor oil, apolyoxyethylene sorbitan fatty acid ester (polysorbates), a poloxamer, avitamin E derivative, a polyoxyethylene alkyl ether, a polyoxyethylenesterate, or saturated polyglycolyzed glyceride or combinations thereof.

In a particular embodiment, the composition is disposed on the articleof manufacture in the form of a coating. In a particular embodiment, thearticle of manufacture comprises a vessel, wherein the composition isdisposed within the vessel. In a particular embodiment, the article ofmanufacture is selected from the group consisting of a fabric article, adiaper, a wound dressing, a medical device, a needle or plurality ofneedles, a microneedle or plurality of microneedles, an injection deviceand a spray dispenser. In a particular embodiment, the article ofmanufacture comprises a plurality of microneedles. In particularembodiments, the medical device is selected from the group consisting ofa prosthetic, an artificial organ or component thereof, a valve, acatheter, a tube, a stent, an artificial membrane, a pacemaker, asensor, an endoscope, an imaging device, a pump, a wire and an implant.In a particular embodiment, the implant is selected from the groupconsisting of a cardiac implant, a cochlear implant, a corneal implant,a cranial implant, a dental implant, a maxillofacial implant, an organimplant, an orthopedic implant, a vascular implant, an intraarticularimplant and a breast implant.

In a particular embodiment, the composition is suitable foradministration by a means selected from the group consisting ofelectroporation, sonication, radio frequency, pressurized spray andcombinations thereof.

In a particular embodiment, the composition is for treating impairedneurological function. In a particular embodiment, the impairedneurological function comprises a decrease in a function selected fromthe group consisting of cognitive function, sensory function, motorfunction and combinations thereof. In particular embodiments, theimpaired neurological function is associated with a condition ordisease, including for example, impaired neurological function isassociated with a condition or disease, including for example, trauma,vascular dementia, senile dementia, Alzheimer's disease, amyotrophiclateral sclerosis (ALS), multiple sclerosis, Parkinson's disease,stroke, schizophrenia, bipolar disorder, depression, obesity, anorexia,cachexia an infection, and an immunological disorder. In a particularembodiment, the impaired neurological function is due to exposure to adrug, such as an anesthetic.

In a particular embodiment, the composition is for treating a skin orscalp disorder selected from the group consisting of alopecia, eczema,psoriasis, seborrheic keratosis and seborrhea. Skin and scalp disclosersinclude disorders of skin, scalp and hair appendages, including forexample, nails and hair follicles. In a particular embodiment, the skindisorder is a skin wound, including for example, a venous leg ulcer, apressure ulcer, a diabetic foot ulcer, a burn, an amputation wound, adecubitus ulcer (bed sore), a split-skin donor graft, a skin graft donorsite, a medical device implantation site, a bite wound, a frostbitewound, a puncture wound, a shrapnel wound, a dermabrasion, a contusion,an infection, a wound and a surgical wound.

In a particular embodiment, the composition is for inducing or promotingtissue tissue repair. As used herein, tissue repair encompassesinduction and promotion of tissue regeneration, including of neuraltissues.

In a particular embodiment, the composition is for inducing or promotingtissue tissue repair following an injury or insult. In a particularembodiment, the injury or insult is selected from the group consistingof a myocardial infarction, a pulmonary embolism, a cerebral infarction,peripheral artery occlusive disease, a hernia, a splenic infarction, avenous ulcer, an axotomy, a retinal detachment, an infection and asurgical procedure.

It is to be understood explicitly that the scope of the presentinvention encompasses shorter and longer forms of polymeric myrcene,including synthetic and semi-synthetic forms, including myrcenecopolymers, and derivatives substituted with various functionalities,and conjugates with additional molecules, as are known in the art, withthe stipulation that these variants and modifications preserve thetherapeutic capacity of the polymeric myrcene in the context of themethods of the present invention.

Other objects, features and advantages of the present invention willbecome clear from the following description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows size exclusion chromatography of a mastic resin extractusing SEDEX and PDA detectors.

FIGS. 2A-B show low (FIG. 2A) and heavy (FIG. 2B) molecular weightfractions of a mastic resin extract obtained by preparative sizeexclusion chromatography.

FIG. 3 shows the ¹³C-NMR spectrum of the heavy MW fraction obtained bypreparative SEC of a mastic resin extract.

FIG. 4 shows the ¹³C-NMR spectrum of the heavy MW fraction obtained bypreparative size exclusion chromatography of a mastic resin extract.

FIGS. 5A-B shows analytical size exclusion chromatography of high (FIG.5A) and low (FIG. 5B) products obtained in a chemical synthetic processfor polymeric myrcene.

FIG. 6 shows the ¹³C-NMR spectrum of the synthesized 1,4-poly-β-myrcene.

FIG. 7 shows the ¹³C-NMR spectrum of the synthesized 1,4-poly-β-myrcene.

FIGS. 8A-D show the effects of RPh-1 on ARPE-19 cells. FIG. 8A, controlcultures treated with oil vehicle; FIG. 8B, test cultures 48 hours afterRPh-1 (0.1%; 1 mg/ml) administration and incubation; FIG. 8C, testcultures 4872 hours after RPh-1 (0.25%; 2.5 mg/ml) administration andincubation; FIG. 8D test cultures 72 hours after RPh-1 (0.25%; 2.5mg/ml) administration and incubation.

FIG. 9 shows immunofluorescence analysis of differentiated ARPE-19 cellsbefore (left panels) and after (right) 72 hours of incubation withRPh-1, indicating expression of tubulin, beta 3 (TUBB3),activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) andneuronal pentraxin II (NPTX2) following the treatment.

FIG. 10 shows the effect of RPh-1 on ARPE-19 cell proliferation asmonitored by an assay to assess total protein content.

FIGS. 11A-C show ARPE-19 cells of various grades of differentiation.FIG. 11A, differentiation grade 3; FIG. 11B, differentiation grade 4;FIG. 11C, differentiation grade 5.

FIGS. 12A-D show the effect of RPh-1 on human melanoma cells. FIG. 12A,oil vehicle treated control cells; FIG. 12B, cells treated with RPh-1 (5μL) after 48 hours incubation, FIG. 12C, cells treated with RPh-1 (2 μL)after 48 hours incubation; FIG. 12D, cells treated with RPh-1 (5 μL)after 72 hours incubation.

FIGS. 13A-B show the effects of chemically synthesized polymeric myrceneon RPh-1 cells. FIG. 13A, differentiation induced with Fraction 18.1;FIG. 13B, differentiation induced with Fraction 18.2.

FIGS. 14A-B shows regeneration of fur in an aging Golden Retriever maledog afflicted with a dermal lesion associated with alopecia followingtreatment with RPh-1. FIG. 14A, prior to treatment; FIG. 14B, following2 weeks of treatment.

FIG. 15 shows the effect of RPh-1 on wound healing of inflicted woundsin experimental mice as indicated by the wound size (mm²) at varioustime points after wound infliction in mice treated with RPh-1 by SCinjection (Group A, grey bars), topically (Group B, black bars) and inmice treated with vehicle alone (Group C, open bars).

FIGS. 16A-G show the effect of RPh-1 on recovery from cerebralhypoperfusion in a vascular dementia rat model, as assessed by theMorris water maze test.

Performance of RPh-1-treated animals (Group A; cross-hatched bars),vehicle treated animals (Group B; horizontally striped bars) and in shamcontrol animals (filled bars) were tested for frequency in platformlocation (FIG. 16A); the time spent in platform area (FIG. 16B); thelatency to find the platform (FIG. 16C); the frequency in zone 1location (FIG. 16D); the time spent in light part (FIG. 16E); thelatency to find the platform (FIG. 16F); and the velocity (FIG. 16G).

FIGS. 17A-B show the effect of RPh-1 on weight gain:

FIG. 17A shows weight gain in animals after cerebral hypoperfusion in avascular dementia rat model. Weight of Group B animals (RPh-1 treated;triangle symbols) is recovering significantly faster then Group Aanimals (vehicle treated; square symbols).

FIG. 17B shows weight gain of obese mice (ob/ob) following treatmentwith RPh-1, either by subcutaneous administration (Group A; diamondsymbols) or by topical administration (Group B; square symbols), ortreatment with vehicle alone (Group C; triangle symbols). Mice of GroupSB and C gained 10.2% and 9.1% respectively. The rate of body weight gainin all groups as expressed by the slopes was similar (p=0.07 (A vs. B),0.08 (A vs. C) and 0.43 (B vs. C).

FIGS. 18A-C show the effect of RPh-1 on recovery from transient middlecerebral artery occlusion (tMCAO) in a rat stroke model:

FIG. 18A shows neuro-muscular score (Neuroscore) at various time pointsin days (d) as indicated, following MCAO in rats treated with RPh-1(Group A) or with vehicle (Group B). Significant differences were seenonly in Group A, between day 8 and day 14, and between day 8 and day 28.

FIG. 18B shows the results of stepping test at various time pointsfollowing MCAO in rats treated with RPh-1 (Group A; black bars) or withvehicle (Group B; open bars) treatment. Significant differences werefound between the two groups only on day 28.

FIG. 18C shows the results of adhesive removal test at various timepoints in days (d) as indicated, following MCAO in rats treated withRPh-1 (Group A) or with vehicle (Group B). Significant differences wereseen only in Group A, between day 2 and the other days.

FIG. 19 shows the average number of surviving Retinal Ganglion Cells(RGC) following axotomy of the optic nerve in RPh-1 treated andcontrol-treated rats.

FIGS. 20A-B shows Western blot analysis of expression of SEMA3 (FIG.20A) and caspase-3 (FIG. 20B) in detached retinas (RD) and non-injuredretinas (control) from animals treated with RPh-1 or vehicle followingretinal detachment.

DETAILED DESCRIPTION OF THE INVENTION

The inventor of the present invention has surprisingly found thatisolated fractions of mastic gum have activity in ameliorating impairedneurological function, healing of skin and scalp disorders and wounds,and in promoting tissue repair. Such fractions are known to containpolymeric myrcene. Furthermore, it has also been surprisingly found thatpurified fractions of polymeric myrcene exhibit the same biologicalactivities as that observed with isolated fractions of mastic gum. Theaforementioned biological activities of polymeric myrcene have beendemonstrated both with that derived from a plant source and thatchemically synthesized. Moreover, the molecular weight range of thepolymer, and the degree of purity of the preparation are importantfactors influencing the biological activity of the polymeric myrcene.These findings are highly unexpected in light of prior art which teachesthat the polymeric fraction obtained from mastic, has no therapeuticbenefit, and in fact hinders certain biological activities attributed tocrude mastic preparations and mastic extracts.

It is herein disclosed for the first time that owing to its variousactivities in stimulating and inducing cell regeneration, the isolatedfraction of mastic gum as described herein may be employed as an activeingredient in a pharmaceutical composition for a number of therapeuticindications

Advantageously, the compositions of the invention may be used in methodsof treating impaired neurological function and skin and scalpconditions. Upon contact with cells of both human and non-humansubjects, the composition induces cell differentiation in a wide arrayof tissues, cell compartments and cell lineages, including skin,endothelium, mucous membranes, bones, tendons and cartilage. Inaddition, the cell differentiation activity of the pharmaceuticalcomposition may be exploited for promoting in vivo incorporation ofmedical devices, implants and organ transplants.

Definitions

As used herein, the terms “mastic”, “mastic resin”, “gum mastic” and“mastic gum”, are used interchangeably to refer to a tree resin (alsoknown as an oleoresin) obtained as an exudate from any tree classifiedin the family Anacardiaceae. Trees in the genus Pistacia, most notablyPistacia lentiscus L., and in particular the cultivar P. lentiscus L.cv. Chia (cultivated on the Greek island of Chios), are known for theirhigh yield of mastic. Other varieties include P. lentiscus L. var.emarginata Engl., and P. lentiscus L. var. latifolia Coss. Additionalspecies of Pistacia include for example, P. atlantica, P. palestina, P.saportae, P. terebinthus, P. vera and P. integerrima.

As used herein, the term “polymer” refers to a compound or a mixture ofcompounds, comprising repeating subunits (also referred to as monomers)of the same chemical structure, wherein the monomers are in covalentconnection. An example of a monomer from which a polymer may be formedis a terpene, for example a monoterpene such as myrcene. Polymers mayhave various degrees of polymerization and thus encompass polymericforms of various chain length. Polymers include homopolymers andheteropolymers (also known as copolymers), and may have various isomericand diastereoisomeric configurations.

As used herein, the terms “polymeric myrcene” and “polymyrcene”interchangeably refer to a polymer formed from myrcene monomers.Polymeric myrcene encompasses polymeric forms having various degrees ofpolymerization and preferably myrcene polymers having a degree ofpolymerization of at least 6. The invention encompasses withoutlimitation, polymeric β-myrcene (poly-β-myrcene), polymeric α-myrcene(poly-α-myrcene), homopolymers thereof, heteropolymers (also known ascopolymers) comprising myrcene monomers in direct or indirect covalentconnection with heterologous monomers, trans- and cis-isomers thereof,D- and L-enantiomers thereof, or combinations thereof. Polymeric myrcenemay be obtained in isolated form from a plant source, in particular frommastic, or may be the product of a chemical synthesis reaction.

As used herein, the term “an isolated fraction of mastic gum” refers toa fraction obtained following extraction of gum mastic in at least onepolar or non-polar organic solvent, or combinations thereof. Theisolated fraction of the invention is generally soluble in either orboth of polar and non-polar organic solvents.

As used herein, the term “an isolated fraction of polymeric myrcene”refers to a preparation of polymeric myrcene having a defined molecularweight or molecular weight range, which is separated away from otherchemical components present in the source from which the polymericmyrcene was isolated, in particular a chemical reaction mixture or aplant extract.

As used herein, the term “degree of purity” refers to the content of aspecified chemical compound in a preparation, expressed as a percentageon a weight per weight basis of the specified chemical compound relativeto other chemical compounds in the preparation.

As used herein, “homopolymer” refers to a polymer that is produced froma single type of monomer. For example, polymeric myrcene is ahomopolymer when it is produced only from myrcene monomers, for exampleβ-myrcene. A homopolymer may also be a mixture of polymers produced fromthe same monomer, but having a varying degree of polymerization i.e.chain length. Accordingly, polymeric myrcene may encompass a range ofcompounds of different chain lengths and accordingly different molecularweights. Further, a homopolymer may contain monomers having differentisomeric configurations, for example, β-myrcene and α-myrcene.

As used herein, “heteropolymer” and “copolymer” refer to a polymerproduced from more than one type of monomer. Thus for example, a myrcenecopolymer is produced from myrcene monomers, in addition to aheterologous type of monomer that is not myrcene. Copolymers includealternating copolymers, periodic copolymers, random copolymers, blockcopolymers and statistical copolymers, as is known in the art.

As used herein, “degree of polymerization” refers to the number ofmonomers or monomeric units which are covalently associated together toform a polymer, for example, the number of myrcene monomers in apolymeric myrcene compound.

As used herein, “weight average molecular weight” refers to the averagemolecular weight of a polymer having molecules of different chainlengths, as expressed by the equation:

${\overset{\_}{M}}_{w} = \frac{\sum\limits_{i}\; {N_{i}M_{i}^{2}}}{\sum\limits_{i}\; {N_{i}M_{i}}}$

where N_(i) is the number of molecules of molecular weight M_(i). Theweight average molecular weight can be determined for example, by lightscattering, small angle neutron scattering, X-ray scattering, andsedimentation velocity.

As used herein, “number average molecular weight” refers to the averagemolecular weight of a polymer having molecules of different chainlengths, as expressed by the equation:

${\overset{\_}{M}}_{n} = \frac{\sum\limits_{i}\; {N_{i}M_{i}}}{\sum\limits_{i}\; N_{i}}$

where N_(i) is the number of molecules of molecular weight M_(i). Thenumber average molecular weight can be determined for example, by gelpermeation chromatography (also known as size exclusion chromatography)or viscometry.

The terms “polydispersity index” and “molecular distribution” are hereinused interchangeably to refer to the ratio of the weight averagemolecular weight to the number average molecular weight.

As used herein, “terpene compounds” refers to isoprene-containinghydrocarbons and related oxygen-containing compounds such as alcohols,aldehydes or ketones (terpenoids). The isoprene unit (CH₂═C(CH₃)—CH═CH₂)is the basic building block of such compounds. Terpene hydrocarbons ingeneral, have the molecular formula (C₅H₈)_(n), and includemonoterpenes, sesquiterpenes, diterpenes, triterpenes, and tetraterpeneswhich respectively have 2, 3, 4, 6 and 8 isoprene units. Terpenes may befurther classified as acyclic or cyclic.

Examples of monoterpenes include myrcene, limonene and pinene, which arerespectively examples of acyclic, monocyclic and bicyclic monoterpenes.Examples of sesquiterpenes include nerolidol and farnesol. Examples ofditerpenes include cafestol and phytol. Examples of a triterpene and atetraterpene are squalene and carotene, respectively.

As used herein, “substantially devoid” means that a preparation orpharmaceutical composition according to the invention that generallycontains less than 3% of the stated substance, preferable less than 1%and most preferably less than 0.5%.

As used herein, “therapeutically effective amount” refers to that amountof a pharmaceutical ingredient which substantially induces, promotes orresults in a desired therapeutic effect.

As used herein, “pharmaceutically acceptable carrier” refers to adiluent or vehicle which is used to enhance the delivery and/orpharmacokinetic properties of a pharmaceutical ingredient with which itis formulated, but has no therapeutic effect of its own, nor does itinduce or cause any undesirable or untoward effect or adverse reactionin the subject.

As used herein, “pharmaceutically acceptable hydrophobic carrier” refersto a hydrophobic non-polar diluent or vehicle in which the polymericmyrcene is dissolved or suspended.

As used herein, “cell differentiation” refers to the process in which aless specialized cell becomes a more specialized cell. Celldifferentiation may be established on the basis of changes in any of anumber of cellular characteristics, including but not limited to size,shape, organelle appearance, membrane potential, metabolic activity, andresponsiveness to signals. A particular “grade” may be given to a celltype to describe the extent of differentiation.

As used herein, “impaired neurological function” refers to a decline ordecrease in at least one of sensory, cognitive or motor function, ascompared to a previous level of function or activity, and/or as comparedto non-impaired individuals matched according to accepted criteria.

Numerical values stated herein are to be understood as the stated value+/−10%.

Isolated Fractions of Mastic Gum and Polymeric Myrcene

The present invention employs isolated fractions comprising polymericmyrcene. The fraction may be from a plant source, in particular masticgum, or it may be the product of a chemical synthesis. Polymeric myrcenefor use in the invention is a polymer compound, or a mixture of polymersof different molecular weights, which are formed from myrcene subunits.Suitable plant sources of polymeric myrcene includes those classifiedeither in the family Anacardiaceae or a different plant family Plantspecies useful for obtaining the compositions of the invention includewithout limitation, those of the genera Pistacia, Pinus, Picea,Juniperus, Alsies, Larix, Ocimum, Laurus and Lavendula. Useful speciesof Pistacia include without limitation, P. lentiscus, P. atlantica, P.palestina, P. saportae, P. terebinthus, P. vera and P. integerrima. Thepolymeric myrcene may be obtained from any plant part, including forexample, resin, leaves, branches, berries and seeds.

An isolated fraction of polymeric myrcene may be most convenientlyobtained from mastic gum, although other plant parts and products may beused. Various methods for obtaining and characterizing an isolatedfraction comprising polymeric myrcene from mastic gum are exemplified inExamples 1 and 2 herein. Commercial preparations of mastic are availablefor example, from the Chios Gum Mastic Growers Association, or from G.Baldwin & Co., U.K.

Alternately, polymeric myrcene may be chemically produced as a syntheticequivalent of a naturally occurring polymer, such ascis-1,4-poly-β-myrcene, or it may be a myrcene polymer not known tooccur in nature, such as polymeric α-myrcene. The invention is notlimited to the process by which the polymeric myrcene is produced orwhether it is natural, synthetic or semi-synthetic.

It is envisioned that the polymeric myrcene may be a synthetic product,produced by a chemical process using as a substrate a monomeric form ofthe monoterpene myrcene. The monomeric myrcene substrate may be isolatedfrom a plant, or may be chemically or enzymatically converted from aprecursor terpene, as is known in the art. For example, monomericβ-myrcene isolated from a plant source may be subsequently polymerizedto polymeric β-myrcene by a chemical process. When the myrcene substrateis derived from a natural source, the resultant product may be referredto as a semi-synthetic product. Chemical processes for polymerizingβ-myrcene are disclosed for example in U.S. Pat. Nos. 4,564,718;5,759,569; 7,232,872 and 7,214,750, and in Newmark et al (1988) J.Polymer Sci. 26, 71-77 (1988) and in Cawse et al (1986) Journal ofApplied Polymer Science, Vol. 31, 1963-1975.

A suitable chemical synthetic process employs an anionic polymerizationreaction, for example that which comprises use of at least one alkane orcycloalkane solvent and at least one alkyl alkali metal. For example,the alkyl alkali metal may be butyl lithium, and the alkane solvent maybe hexane, or the cycloalkane solvent may be cyclohexane. The alkanesolvent and the alkyl alkali metal initiator may be present in thereaction mixture at a ratio of at least 20:1. The anionic polymerizationreaction may be terminated by a compound such as water, an alcohol,molecular oxygen and carbon dioxide.

The synthetic process for 1,4-poly-β-myrcene disclosed herein (Example3) is particularly suitable for maintaining the various biologicalactivities of the polymer, such as promoting cell differentiation.Monomeric β-myrcene is known to occur in a variety of plants, includingtrees in the genera Pinus, Picea, Juniperus, Alsies and Larix, andflowers in the genera Antirrhinum, Boswellia, Citrus and Gynura.

An isolated fraction of polymeric myrcene may be obtained as thepurified product of a chemical synthesis reaction, as exemplified inExample 3 herein. Chemically synthesized polymeric myrcene may beisolated from unreacted substrate and other reagents, analyzed andfurther fractionated according to molecular weight using analytical andseparation methods as are known in the art. Such methods include thosewhich separate molecules on the basis of size, charge or hydrophobicity,including for example, size exclusion chromatography (SEC), highpressure liquid chromatography (HPLC), gas liquid chromatography (GLC)and combinations thereof.

Analytical methods for determining the precise chemical structure of theobtained polymer include nuclear magnetic resonance (for example ¹HNMRand ¹³CNMR), viscometry, various mass spectrometry methods (for exampleMALDI-TOF), combination methods such as Liquid Chromatography-Massspectrometry (LC-MS)), light-scattering techniques such as for exampleMulti Angle Laser Light Scattering (MALLS), total carbon analysis,UV-VIS spectrophotometry, IR and FT-IR spectrophotometry and othermethods as are known in the art. The same methods and approaches may beused for purifying and characterizing polymeric myrcene from plants, asshown herein in Example 2.

In a particularly preferred embodiment, a fraction of polymeric myrcenewhich is a product of a chemical synthesis should be substantiallydevoid of myrcene monomers and myrcene oligomeric forms having a degreeof polymerization less than about 5. It is also preferred that theisolated product be substantially devoid of monomeric terpene compoundswhich are soluble in polar organic solvents.

Similar methods may be used for obtaining isolated fractions of masticgum and isolated fractions of polymeric myrcene, when the polymericmyrcene is to be derived from a plant source, such as mastic gum. By wayof a general description, collected plant material, for example masticgum, is combined in a suitable vessel with a suitable solvent, usually apolar solvent. Suitable polar solvents include for example, alcohols,ethers, esters, amides, aldehydes, ketones, nitriles and combinationsthereof. Particular examples of polar organic solvents are methanol,ethanol, propanol, isopropanol, 1-butanol, 2-butanol, sec-butanol,t-butanol, 1-pentanol, 2-pentanol, 3-pentanol, neopentanol,3-methyl-1-butanol, 2-methyl-1-butanol, 3-methyl-2-butanol,2-methyl-2-butanol, ethyleneglycol, ethyleneglycol monomethyl ether,diethyl ether, methylethyl ether, ethylpropyl ether, methylpropyl ether,1,2-dimethoxyethane, tetrahydrofuran, dihydrofuran, furan, pyran,dihydropyran, tetrahydropyran, methyl acetate, ethyl acetate, propylacetate, acetaldehyde, methylformate, ethylformate, ethyl propionate,methyl propionate, dichloromethane, chloroform, dimethylformamide,acetamide, dimethylacetamide, N-methylpyrrolidone, acetone, ethylmethylketone, diethyl ketone, acetonitrile, propionitrile, and combinationsthereof.

The mastic gum and the solvent are preferably combined such that thesolvent is in large excess, for example 10:1 or 20:1. The mixture may beperiodically or continuously agitated over a period ranging from a fewminutes to a number of hours. The solvent may be decanted without anytreatment, or optionally the mixture may be first subjected to low speedcentrifugation, for example at 100 to 2000 rpm, as is known in the art.The insoluble material is recovered from the extract and a fresh aliquotof solvent is added to the insoluble material, such that the extractionand dissolution process is repeated for a number of cycles, in order toobtain as much as possible of the polar solvent soluble compounds. Afterthe final dissolution step, the extracts containing polar solventsoluble material are combined and the polar solvent is evaporated (forexample by using a rotary evaporation as is known in the art), so as toyield polar solvent soluble material, which may be referred to as acrude, or “first step” extract.

The first step extract material is combined with a non-polar organicsolvent and extracted by shaking over a period of 1 hour. Suitablenon-polar solvents include acyclic or cyclic, saturated or unsaturatedaliphatic hydrocarbons and aromatic hydrocarbons, for example, C5-C10alkanes, C5-C10 cycloalkanes, C6-C14 aromatic hydrocarbons, andcombinations thereof. Each of the foregoing may be optionallysubstituted by one or more halogens, for example, C7-C14perfluoroalkanes. Particular examples of non-polar organic solvents arepentanes, hexanes, heptanes, octanes, nonanes, decanes, cyclopentane,cyclohexane, cycloheptane, benzene, toluene, xylene, and isomers andmixtures thereof. Material remaining insoluble or precipitating in thepresence of the non-polar solvent is removed and discarded. Thenon-polar solvent-soluble fraction is then obtained by evaporating thenon-polar solvent (for example by rotary evaporation). This fraction maybe referred to as purified or “two step” extract, corresponding to anisolated fraction of mastic gum which is characterized by the fact thatit is soluble in both a polar solvent and a non-polar solvent, whilematerials which are soluble in the polar solvent but insoluble in thenon-polar solvent, have been removed. This feature distinguishes theisolated fractions of the invention over prior art extracts of masticgum, the latter of which generally include a wide variety of compoundswhich are soluble only in polar solvents. According to the teachings ofthe present invention, such compounds interfere with the beneficialbiological activities of the isolated fractions disclosed herein.

The two step extract may be dried further, for example by high vacuumtreatment (for example <0.01 mbar for up to several days) to removeresidual solvent and other volatile material, weighed and combined witha suitable non-polar organic solvent or other carrier to effect itsdissolution. As disclosed herein in Examples 1 and 2, such isolatedfractions contain polymeric myrcene. The obtained fractions containingpolymeric myrcene may be used directly, or further purified,characterized and/or fractionated using means known in the art, asenumerated above.

In particular embodiments, the isolated fractions of the invention maybe obtained by a process comprising the steps of:

-   -   (a) treating mastic gum with a polar organic solvent;    -   (b) isolating a fraction soluble in said polar organic solvent;    -   (c) optionally removing said polar organic solvent;    -   (d) treating the soluble fraction obtained in step (b) or (c)        with a non-polar organic solvent, (e) isolating a fraction        soluble in said nonpolar organic solvent; and    -   (f) optionally removing said nonpolar organic solvent;

wherein steps (d) to (f) may precede steps (a) to (c).

The process may further comprise size fractionating the soluble fractionobtained following step (c) or step (f), for example by size exclusionchromatography, or any other method known in the art.

The process may further comprise removing the solvent after either orboth of steps (c) or (f). Solvent removal may be carried out by anymeans known in the art, for example rotary evaporation, application ofhigh vacuum and a combination thereof. In particular embodiments, steps(a) to (c) are carried out prior to steps (d) to (f) or vice versa. In aparticular embodiment, the polar organic solvent comprises ethanol andthe non-polar organic solvent comprises hexane. As is readily understoodby one of skill in the art, steps (a) to (c) and steps (d) to (f) mayeach be independently carried out for a number of cycles to optimize theextraction process and degree of purification of the product.

For preparation of a composition for therapeutic use, suitable carriersmay be used, such as hydrophobic carriers including pharmaceuticallyacceptable oils, optionally in combination with waxes, as describedherein.

In particularly preferred embodiments, the compositions comprising thefractions isolated from mastic gum as herein described, should compriseless than about 20% (w/w) of monomeric and oligomeric terpene compoundswhich are soluble in the polar organic solvent and are substantiallyinsoluble in the non-polar organic solvent, wherein the aforementionedsolvents refer to those used in the preparation of the fraction. Morepreferably, the isolated fractions comprise less than about 5% (w/w) ofsuch terpene compounds. Even more preferably, the isolated fractions aresubstantially devoid of such terpene compounds. The inhibitory effectsof fractions comprising such low molecular weight compounds on thebiological activity of polymeric myrcene are exemplified herein inExample 8.

In another particular embodiment, an isolated fraction comprisingpolymeric myrcene is derived from a plant and is substantially devoid ofmyrcene monomers and myrcene oligomeric forms having a degree ofpolymerization less than 6. In another particular embodiment, anisolated fraction comprising polymeric myrcene is derived from a plantand is substantially devoid of terpene compounds which are soluble in apolar organic solvent but are substantially insoluble in a non-polarorganic solvent.

It is to be understood that the polymeric myrcene may not have a singlemolecular weight, but rather, a distribution of molecular weights,representing a population of polymeric myrcene molecules of differentchain length i.e. degree of polymerization.

There is no particular upper limit on the molecular weight or degree ofpolymerization of the polymeric myrcene. In one currently preferredembodiment of the invention, the degree of polymerization is at leastabout 6. In a particular embodiment, the degree of polymerization is atleast about 10. In a particular embodiment, the degree of polymerizationis at least about 25. In a particular embodiment, the degree ofpolymerization is at least about 35. In a particular embodiment, thepolymeric myrcene has a degree of polymerization in the range of atleast about 6 to about 1800. Suitable exemplary ranges include about 30to about 500, or about 35 to about 150. The number average molecularweight of the polymeric myrcene is preferably at least about 800. Morepreferably, the number average molecular weight is at least about 1,000,such as at least 2000 or at least 3000, and even more preferably, thenumber average molecular weight is at least about 5000. In a particularembodiment, the polymeric myrcene has a number average molecular weightin the range from about 5000 to about 20,000. In a particularembodiment, the polymeric myrcene has a number average molecular weightin the range from at least about 800 to about 100,000. In particularembodiments, the number average molecular weight is in a range selectedfrom the group consisting of: at least about 800 to about 5000; at leastabout 800 to about 15,000; about 5000 to about 15,000; about 5000 toabout 20,000; about 15,000 to about 30,000; about 25,000 to about40,000; about 35,000 to about 50,000; about 45,000 to about 60,000;about 55,000 to about 70,000; about 65,000 to about 80,000; about 75,000to about 90,000; about 85,000 to about 100,000; and combinationsthereof. In a particular embodiment, the number average molecular weightis at least about 5000. It is to be understood that the composition maycomprise different molecular weight fractions of polymeric myrcene, forexample in the range from at least about 5000 to about 20,000, as wellas in the range from about 25,000 to about 40,000. In a particularembodiment, the polymeric myrcene has a molecular distribution of lessthan 5.

In a particular embodiment, the isolated fraction consists essentiallyof polymeric myrcene that has a number average molecular weight in therange from about 5000 to about 20,000.

The molecular weight of the polymeric product may be expressed in anumber of ways, for example, weight average molecular weight or numberaverage molecular weight, as is known in the art. Molecular weight maybe determined by any of a number of means, such as light scattering,multi angle laser light scattering (MALLS), small angle neutronscattering, X-ray scattering, sedimentation velocity, viscometry(Mark-Houwink equation), mass spectrometry (e.g. MALDI-TOF) and gelpermeation chromatography.

The polymeric myrcene may exist as different geometric isomers,resulting from the arrangement of substituents around the carbon-carbondouble bond. Such isomers are designated as the cis- ortrans-configuration (also referred to respectively as the Z or Econfiguration), wherein cis- (or Z) represents substituents on the sameside of the carbon-carbon double bond, and trans- (or E) representssubstituents on opposite sides of the carbon-carbon double bond. Thevarious geometric isomers and mixtures thereof are included within thescope of the invention.

The polymeric myrcene product may contain one or more asymmetric carbonatoms and may therefore exhibit optical isomerism and/ordiastereoisomerism. All stereoisomers and diastereoisomers are includedwithin the scope of the invention, either as a single isomer or as amixture of sterochemical isomeric forms. The various stereoisomers anddiastereoisomers may be separated using conventional techniques, forexample chromatography or fractional crystallisation. Alternativelydesired optical isomers may be made by reaction of the appropriateoptically active starting materials under conditions which will notcause racemisation or epimerisation, or by derivatisation, for examplewith a homochiral acid followed by separation of the diastereomericderivatives by conventional means.

Suitable forms of polymeric myrcene include polymeric β-myrcene(poly-β-myrcene), including 1,4-poly-β-myrcene, 3,4-poly-β-myrcene,1,2-poly-β-myrcene, cis-1,4-poly-β-myrcene, trans-1,4-poly-β-myrcene,polymeric α-myrcene (poly-α-myrcene) or combinations thereof. Theisolation and characterization of 1,4-poly-β-myrcene from mastic isdisclosed for example in Van der Berg et al (1998) Tetrahedron Lett3:2645-2648.

In particular embodiments, the polymeric myrcene has a linearconformation, a branched conformation or a cyclic conformation.

The isolated fraction of polymeric myrcene according to the inventionhas a degree of purity of at least 90%, such as at least 93%, or atleast 95%, or at least 97%, or at least 98% or at least 99%. As isunderstood in the art, as high a degree of purity as possible isdesirable inter alia to ensure compliance with health regulatory agencyrequirements. It is to be understood however, that the fraction ofpolymeric myrcene may contain myrcene polymeric species having variousmolecular weights, such as within a defined narrow or wide range,without reducing the specified degree of purity. In addition, theisolated fraction of polymeric myrcene may contain different structuralisomers as described above of polymeric myrcene without reducing thespecified degree of purity. In a particular embodiment, the isolatedfraction of polymeric myrcene comprises at least 90% (w/w) ofcis-1,4-poly-β-myrcene. In a particular embodiment, the isolatedfraction of polymeric myrcene comprises a mixture ofcis-1,4-poly-β-myrcene and trans-1,4-poly-β-myrcene, wherein the mixturecomprises at least 80% (w/w) of cis-1,4-poly-β-myrcene. In a particularembodiment, the isolated fraction of polymeric myrcene comprises atleast 90% (w/w) of cis-1,4-poly-β-myrcene having a number averagemolecular weight of at least 800. The number average molecular weightmay be at least 1,000. The average molecular weight may be at least2000. The number average molecular weight may be at least 3,000. Thenumber average molecular weight may be at least 5000. The number averagemolecular weight may be at least 10,000. In a particular embodiment, theisolated fraction of polymeric myrcene comprises at least 90% (w/w) ofcis-1,4-poly-β-myrcene having a number average molecular weight in therange from about 800 to about 5000. In a particular embodiment, theisolated fraction of polymeric myrcene comprises at least 90% (w/w) ofcis-1,4-poly-β-myrcene having a number average molecular weight in therange from about 1,000 to about 10,000. In a particular embodiment, theisolated fraction of polymeric myrcene comprises at least 90% (w/w) ofcis-1,4-poly-β-myrcene having a number average molecular weight in therange from about 10,000 to about 20,000. In a particular embodiment, theisolated fraction of polymeric myrcene comprises at least 90% (w/w) ofcis-1,4-poly-β-myrcene having a number average molecular weight in therange from about 5000 to about 20,000. In a particular embodiment, theisolated fraction of polymeric myrcene consists essentially ofcis-1,4-poly-β-myrcene that has a number average molecular weight in therange from about 5000 to about 20,000.

In a particular embodiment, the isolated fraction of polymeric myrcenehas a degree of purity of at least 90%, and the polymeric myrcene has adegree of polymerization of at least 10.

In a particular embodiment, the isolated fraction of polymeric myrcenecomprises at least 90% (w/w) of cis-1,4-poly-β-myrcene having a numberaverage molecular weight in the range from about 20,000 to about 30,000.In a particular embodiment, the isolated fraction of polymeric myrcenecomprises at least 90% (w/w) of cis-1,4-poly-β-myrcene having a numberaverage molecular weight in the range from about 30,000 to about 50,000.In a particular embodiment, the isolated fraction of polymeric myrcenecomprises at least 90% (w/w) of cis-1,4-poly-β-myrcene having a numberaverage molecular weight in the range from about 50,000 to about 80,000.

In particularly preferred embodiments, the isolated fraction ofpolymeric myrcene is substantially purified of terpene compounds whichare soluble in a polar organic solvent but substantially insoluble in anon-polar organic solvent. In particular, the composition shouldcomprise less than about 10% (w/w), and more preferably, less than about5% (w/w), and most preferably, less than about 3% (w/w), of terpenecompounds which are soluble in a polar organic solvent but substantiallyinsoluble in a non-polar organic solvent. In particular embodiments, thecomposition is substantially devoid of terpene compounds which aresoluble in a polar organic solvent but insoluble in a non-polar organicsolvent. In particular embodiments, the composition comprises less thanabout 10% (w/w), and more preferably less than about 5% (w/w), and mostpreferably, less than about 3% (w/w), of monomeric terpene compounds. Ina particular embodiment, the composition is substantially devoid ofmyrcene monomers and myrcene oligomeric forms having a degree ofpolymerization less than about 5. In a particular embodiment, thecomposition comprises less than about 10% (w/w), and more preferably,less than about 5% (w/w), and most preferably, less than about 3% (w/w),of a terpene compound selected from the group consisting of: β-myrcene,α-myrcene, cis-α-ocimene, dihydromyrcene, limonene, α-pinene, β-pinene,and combinations thereof.

Pharmaceutical Compositions

The composition for use in the invention comprises a therapeuticallyeffective amount of an isolated fraction of polymeric myrcene, and apharmaceutically acceptable hydrophobic carrier.

A suitable hydrophobic carrier comprises at least one oil, such as forexample a mineral oil, a vegetable oil or combinations thereof.

The term “mineral oil” refers to a clear colorless nearly odorless andtasteless liquid obtained from the distillation of petroleum. It mayalso be referred to as white oil, white mineral oil, liquid petrolatum,liquid paraffin or white paraffin oil. In accordance with a particularembodiment of the invention, the mineral oil is light mineral oil, acommercially available product which may be obtained either as a NF(National Formulary) grade product or as a USP (US Pharmacopoeia) gradeproduct. For use in the invention, the mineral oil is preferably free ofaromatics and unsaturated compounds.

Suitable vegetable oils include, but are not limited to almond oil,canola oil, coconut oil, corn oil, cottonseed oil, grape seed oil, oliveoil peanut oil, saffron oil, sesame oil, soybean oil, or combinationsthereof. In a particular embodiment, the mineral oil is light mineraloil.

The pharmaceutically acceptable carrier may alternately or in additioncomprise a suitable oil replacement. Oil replacements include alkaneshaving at least 10 carbon (e.g., isohexadecane), benzoate esters,aliphatic esters, noncomodogenic esters, volatile silicone compounds(e.g., cyclomethicone), and volatile silicone substitutes. Examples ofbenzoate esters include C₁₂C₁₅ alkyl benzoate, isostearyl benzoate,2-ethyl hexyl benzoate, dipropylene glycol benzoate, octyldodecylbenzoate, stearyl benzoate, and behenyl benzoate. Examples of aliphaticesters include C₁₂C₁₅ alkyl octonoate and dioctyl maleate. Examples ofnoncomodogenic esters include isononyl isononanoate, isodecylisononanoate, diisostearyl dimer dilinoleate, arachidyl propionate, andisotridecyl isononanoate. Examples of volatile silicone substitutesinclude isohexyl decanoate, octyl isononanoate, isononyl octanoate, anddiethylene glycol dioctanoate.

Cyclomethicone is an evaporative silicone which may be included in thecarrier to assist in making the composition amenable to ejection from aspray dispenser. Furthermore, due to its evaporative property,cyclomethicone may assist in retaining and fixing the formulation on thesurface to which it is sprayed e.g. a wound site.

The hydrophobic carrier may further comprise at least one wax. Waxesinclude for example, beeswax; vegetable waxes, sugar cane waxes, mineralwaxes, and synthetic waxes. Vegetable waxes include for example,carnauba, candelilla, ouricury and jojoba wax. Mineral waxes include forexample, paraffin wax, lignite wax, microcrystalline waxes andozokerites. Synthetic waxes include for example, polyethylene waxes.

The pharmaceutical composition may be formulated in any of a number offorms such as for example, a capsule (including a softgel capsule), atablet, a gel, a liposome, a suppository, a suspension, an ointment, asolution, an emulsion or microemulsion, a film, a cement, a powder, aglue, an aerosol, a spray and a gel.

For preparing the pharmaceutical composition, the polymeric myrcene maybe suitably formulated as inclusion complexes, nanoemulsions,microemulsions, powders and liposomes. In a particular embodiment, aninclusion complex comprises at least one cyclodextrin. In a particularembodiment, cyclodextrins comprise hydroxypropyl-β-cyclodextrin. In aparticular embodiment, nanoemulsions comprise droplets having averageparticle size of less than 800 nm. In a particular embodiment, thedroplets have average particle size of less than 500 nm. In a particularembodiment, the droplets have average particle size of less than 200 nm.In a particular embodiment, powders are spray dried powders. In aparticular embodiment, liposomes comprise multilamellar vesicles. In aparticular embodiment, a microemulsion comprises a non-ionic surfactant.Non-ionic surfactants include, without limitation, polyoxyl castor oils,polyoxyethylene sorbitan fatty acid esters (polysorbates), a poloxamer,a vitamin E derivative, polyoxyethylene alkyl ethers, polyoxyethylenesterates, saturated polyglycolyzed glycerides or combinations thereof.

Various formulations of polymeric myrcene and preparation thereof aredisclosed herein in Examples 17-21. The pharmaceutical compositions ofthe invention may be administered by any means that achieve theirintended purpose. For example, administration may be by oral,parenteral, topical or transdermal routes. Parenteral administrationincludes intravenous, intramuscular, subcutaneous, intradermal,intraperitoneal, intraarterial, intrauterine, intraurethral,intracardial, intracerebral, intracerebroventricular, intrarenal,intrahepatic, intratendon, intraosseus and intrathecal routes ofadministration. Topical administration includes application via a routeselected from dermal, vaginal, rectal, inhalation, intranasal, ocular,auricular and buccal., The administering may in addition comprise atechnique or means such as electroporation, or sonication in order toassist in their delivery, for example transdermally. Other techniqueswhich may be employed include for example, radio frequency orpressurized spray application.

The dosage administered will be dependent upon the age, health, andweight of the subject, the use of concurrent treatment, if any,frequency of treatment, and the nature of the effect desired. The amountof the polymeric myrcene of the present invention in any unit dosageform comprises a therapeutically effective amount which may varydepending on the recipient subject, route and frequency ofadministration.

In general, the amount of polymeric myrcene or isolated mastic gumfraction present in the pharmaceutical composition may conveniently bein the range from about 0.01% to about 25%, such as 0.01% to about 12%,on a weight per weight basis, based on the total weight of thecomposition. For topical use, the percentage of polymeric myrcene orisolated mastic gum fraction in the composition may be in the range fromabout 0.05% to about 2.5%. For administration by injection, thepercentage of polymeric myrcene or isolated mastic gum fraction in thecomposition may be conveniently in the range from about 0.1% to about7%. For oral administration, the percentage of polymeric myrcene orisolated mastic gum fraction in the composition may be in the range fromabout 0.005% to about 7%.

The pharmaceutical compositions of the invention may be manufactured ina manner which is itself known to one skilled in the art, for example,by means of conventional mixing, granulating, dragee-making, softgelencapsulation, dissolving, extracting, or lyophilizing processes. Inpreferred embodiments, the formulations are non-aqueous and/or do notcomprise polar solvents which directly contact the polymeric myrceneactive ingredient, so as to avoid loss of biological activity of theactive ingredient. Thus, pharmaceutical compositions for oral use may beobtained by combining the active compounds with solid and semi-solidexcipients and suitable preservatives, and/or antioxidants. Optionally,the resulting mixture may be ground and processed. The resulting mixtureof granules may be used, after adding suitable auxiliaries, ifnecessary, to obtain tablets, softgels, capsules, or dragee cores.

Suitable excipients are, in particular, fillers such as saccharides,e.g., lactose or sucrose, mannitol or sorbitol; cellulose preparationsand/or calcium phosphates, e.g., tricalcium phosphate or calciumhydrogen phosphate; as well as binders, such as starch paste, using,e.g., maize starch, wheat starch, rice starch, potato starch, gelatin,tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodiumcarboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired,disintegrating agents may be added such as the above-mentioned starchesand also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar,or alginic acid or a salt thereof, such as sodium alginate. Auxiliariesare flow-regulating agents and lubricants, e.g., silica, talc, stearicacid or salts thereof, such as magnesium stearate or calcium stearate,and/or polyethylene glycol. Dragee cores are provided with suitablecoatings which, if desired, are resistant to gastric juices. For thispurpose, concentrated saccharide solutions may be used, which mayoptionally contain gum arabic, talc, polyvinyl pyrrolidone, polyethyleneglycol and/or titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. In order to produce coatings resistant togastric juices, solutions of suitable cellulose preparations, such asacetylcellulose phthalate or hydroxypropymethyl-cellulose phthalate, areused. Dye stuffs or pigments may be added to the tablets or drageecoatings, e.g., for identification or in order to characterizecombinations of active compound doses.

Other pharmaceutical compositions for oral use include push-fit capsulesmade of gelatin, as well as soft, sealed capsules made of gelatin and aplasticizer, such as glycerol or sorbitol. The push-fit capsules cancontain the active compounds in the form of granules, which may be mixedwith fillers, such as lactose; binders, such as starches; and/orlubricants, such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds are preferablydissolved or suspended in suitable liquids, such as fatty oils, orliquid paraffin. In addition, stabilizers may be added.

Other pharmaceutical compositions for oral use include a film designedto adhere to the oral mucosa, as disclosed for example in U.S. Pat. Nos.4,713,243; 5,948,430; 6,177,096; 6,284,264; 6,592,887, and 6,709,671.

Pharmaceutical compositions in the form of suppositories consist of acombination of the active compound(s) with a suppository base. Suitablesuppository bases include for example, natural or synthetictriglycerides, polyethylene glycols, or paraffin hydrocarbons.

Formulations for parenteral administration include suspensions andmicroparticle dispersions of the active compounds as appropriate. In aparticular embodiment, oily injection suspensions may be administered.Suitable lipophilic solvents or vehicles include fatty oils, e.g.,sesame oil, or synthetic fatty acid esters, e.g., ethyl oleate,triglycerides, polyethylene glycol-400, cremophor, or cyclodextrins.Injection suspensions may contain substances which increase theviscosity of the suspension include, e.g., sodium carboxymethylcellulose, sorbitol, and/or dextran. Optionally, the suspension may alsocontain stabilizers.

Pharmaceutical compositions can also be prepared using liposomescomprising the active ingredient. As is known in the art, liposomes aregenerally derived from phospholipids or other lipid substances.Liposomes are formed by mono- or multi-lamellar hydrated liquid crystalswhich are dispersed in an aqueous medium. Any non-toxic, physiologicallyacceptable and metabolizable lipid capable of forming liposomes can beused. In general, the preferred lipids are phospholipids and thephosphatidyl cholines (lecithins), both natural and synthetic. Methodsto form liposomes are known in the art, as disclosed for example, inPrescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, NewYork, N.Y. (1976) and in U.S. Pat. No. 7,048,943.

Formulations for topical administration include ointments. Suitablecarriers include vegetable or mineral oils, white petrolatum, branchedchain fats or oils, animal fats and waxes. The preferred carriers arethose in which the active ingredient is soluble. Stabilizers, humectantsand antioxidants may also be included, as well as agents imparting coloror fragrance, if desired. Ointments may be formulated for example, bymixing a solution of the active ingredient in a vegetable oil such asalmond oil with warm soft paraffin, and allowing the mixture to cool.

The pharmaceutical composition may comprise an oil-in-water emulsion ormicroemulsion in order to facilitate its formulation for oral,parenteral or topical use Such emulsions/microemulsions generallyinclude lipids, surfactants, optionally humectants, and water. Suitablelipids include those generally know to be useful for creatingoil-in-water emulsions/microemulsions, for example fatty acid glycerideesters. Suitable surfactants include those generally known to be usefulfor creating oil-in-water emulsions/microemulsions wherein lipids areused as the oil component in the emulsion. Non-ionic surfactants may bepreferred, such as for example, ethoxylated castor oil, phospholipids,and block copolymers of ethylene oxide and propylene oxide. Suitablehumectants, if used, include for example propylene glycol orpolyethylene glycol.

The pharmaceutical composition may be formulated in the form of a gel,such as a hydrogel formed from a gel-forming polymer such ascarrageenan, xanthan gum, gum karaya, gum acacia, locust bean gum, guargum. A hydrogel may be combined with an oil-in-water emulsion comprisingthe active ingredient.

The pharmaceutical composition may be formulated in the form of a cementsuch as those comprising polymethylmetacrylate (PMMA) or calciumphosphate, as are used in orthopedic surgery.

The pharmaceutical composition may be formulated in the form of apowder, in particular such as those used for transdermal applicationsusing radio frequency, as described for example, in U.S. Pat. Nos.6,074,688 and 6,319,541 and WO 2006/003659.

The pharmaceutical composition may be formulated in the form of a glue,such as those comprising octocyanoacrylate used for wound closureapplications.

In a particular embodiment, the pharmaceutical composition issubstantially devoid of monomeric and low molecular weight terpenecompounds, including for example, those classified as monoterpenes,diterpenes, sesquiterpenes, triterpenes, tetraterpenes. Examples ofterpene compounds include β-myrcene, α-myrcene, cis-α-ocimene,dihydromyrcene, limonene, α-pinene, β-pinene, tirucallol, betulonal,masticadienonic acid, masticadienolic acid, isomasticadienonic acid,isomasticadienolic acid, oleanolic acid, and oleanonic acid.

Therapeutic Uses

The present invention provides therapeutic uses and methods of treatingimpaired neurological function, treating skin and scalp disorders,inducing tissue repair and wounds in a subject in need thereof. Themethods comprise administering to the subject a therapeuticallyeffective amount of a composition comprising an isolated fraction ofmastic gum, or an isolated fraction of polymeric myrcene, as describedherein.

The step of administering the compositions may comprise any acceptableroute including oral, topical, parenteral, and transdermal. Parenteraladministration includes intravenous, intramuscular, subcutaneous,intradermal, intraperitoneal, intraarterial, intrauterine,intraurethral, intracardial, intracerebral, intracerebroventricular,intrarenal, intrahepatic, intratendon, intraosseus and intrathecalroutes of administration. Topical administration includes applicationvia a route selected from dermal, vaginal, rectal, inhalation,intranasal, ocular, auricular and buccal.

In particular embodiments, the step of administering comprisescontacting cells of a particular type, of a particular lineage or at aparticular stage of differentiation, with the composition. The cells maybe any of a wide variety of cell types, including in particular, neuralcells, neuronal cells, endothelial cells, epithelial cells and stemcells of said lineages. Further, the cells may be of any lineage forexample, ectodermal, mesodermal, entodermal lineages and stem cells ofsaid lineages. In various embodiments, the step of contacting cells iscarried out in vivo, ex vivo or in vitro.

The method disclosed herein for treating impaired neurological functionis particularly advantageous for subjects afflicted withneurodegenerative conditions and diseases, including in particular,trauma, vascular dementia, senile dementia, Alzheimer's disease,amyotrophic laterial sclerosis (ALS), multiple sclerosis), stroke andParkinson's disease. In other cases, the method may be advantageouslyapplied in subjects suffering from impaired neurological function due toan infection (e.g. viral, bacterial, fungal, parasitic) or animmunological disorder. In a particular embodiment, the impairedneurological function is due to exposure to a drug, such as ananesthetic. Impaired neurological function may also be associated with acondition selected from the group consisting of schizophrenia, bipolardisorder, depression, obesity, anorexia and cachexia.

Skin and scalp disorders include all disorders of skin, scalp and hairappendages, including for example, nails and hair follicles. Particularconditions that may benefit from the invention include alopecia, eczema,psoriasis, seborrheic keratosis, seborrhea and skin wounds. Skin woundsinclude venous leg ulcers, pressure ulcers, diabetic foot ulcers, burns,amputation wounds, decubitus ulcers (bed sore), split-skin donor grafts,skin graft donor sites, medical device implantation sites, bite wounds,frostbite wounds, puncture wounds, shrapnel wounds, dermabrasions,contusions, an infection wounds and surgical wounds. Wounds may be theresult of infection; exposure to ionizing radiation; exposure to laser,or exposure to a chemical agent.

The invention may be particularly effective for scar-less repair ofwounds.

The invention may be particularly effective and economical for treatmentof chronic non-healing wounds. As is known to one of ordinary skill inthe art, the efficacy of a particular treatment in promoting woundhealing may be assessed by various criteria, including the rate ofclosure measured by length, width and depth of the wound over time,epithelization rate, formation of granulation tissue and tissue tensilestrength.

The methods disclosed herein for inducing or promoting tissueregeneration are particularly advantageous for subjects who have tissuedamage, which for example, may be associated with, or the result of aninjury or insult. The methods for inducing or promoting tissueregeneration may be used in subjects who have suffered an injury orinsult selected from the group consisting of a myocardial infarction, apulmonary embolism, a cerebral infarction, peripheral artery occlusivedisease, a hernia, a splenic infarction, a venous ulcer, an axotomy, aretinal detachment, a wound (for example, a burn wound, bite wound, afrostbite wound, a puncture wound, a shrapnel wound, a contusion, aninfection wound or a surgical wound), an infection and a surgicalprocedure.

The methods of the invention are exemplified in the Examples disclosedherein Example 4 discloses that an isolated fraction of polymericmyrcene (derived from mastic of Pistacia) induces differentiation ofretinal pigment epithelium cells.

Example 5 discloses that polymeric myrcene shortens the recovery timefrom anesthesia in experimental animals.

Example 6 discloses that the same fraction has activity in inducingdifferentiation in melanoma and neuroblastoma tumor cell lines.

Example 7 discloses that chemically synthesized polymeric myrcene ofvarious molecular weight ranges induces differentiation in retinalpigment epithelium cells.

Example 8 discloses that small molecular weight compounds from masticwhich are separated from polymeric myrcene during preparation thereof onthe basis of their being soluble only in a polar solvent in accordancewith the invention, interfere with, reduce and hinder the celldifferentiation inducing activity exerted by polymeric myrcene.

Examples 9, 10 and 11 disclose that the invention may be applied towound healing in mammals and non-mammalian subjects.

Example 12 discloses that compositions comprising polymeric myrceneaccording to the invention have ameliorating effects in an animal modelof vascular dementia.

Example 13 discloses that the invention may be used to stimulateappetite in subjects affected by various disorders that result inappetite loss or pathological weight gain result in obesity.

Example 14 discloses that compositions comprising polymeric myrceneaccording to the invention have ameliorating effects in an animal modelof stroke.

Example 15 discloses that compositions comprising polymeric myrceneaccording to the invention have ameliorating effects in an animal modelof optic nerve injury/trauma.

Example 16 discloses that compositions comprising polymeric myrceneaccording to the invention have ameliorating effects in an animal modelof retinal detachment and provides evidence of scar-less repair ofwounds. The step of contacting cells may be carried out in vitro or exvivo. In particular, cells, or an organ or tissue derived therefromwhich is intended for implantation or transplantation into the subjectmay be treated according to the invention. For example, cell explants orcells or tissues grown and maintained in culture may be contacted withthe composition. The cells may originate for example, from stem cells ofan autologous or homologous donor, and be intended for organregeneration and/or implantation into a recipient. In other cases, thecells are from a heterologous donor and are intended for implantation ortransplantation into a recipient. In a particular embodiment, the cellsare those of an organ or tissue from a heterologous donor intended forimplantation or transplantation into a recipient. In a particularembodiment, the cells are those which secrete soluble factors.

The method may be carried out prior to or following implantation of amedical device into the subject. Medical devices include, but are notlimited to a prosthetic, an artificial organ or component thereof, avalve, a catheter, a tube, a stent, an artificial membrane, a pacemaker,a sensor, an endoscope, an imaging device, a pump, a wire and animplant. Implants include, but are not limited to a cardiac implant, acochlear implant, a corneal implant, a cranial implant, a dentalimplant, a maxillofacial implant, an organ implant, an orthopedicimplant, a vascular implant, an intraarticular implant and a breastimplant.

In a particular embodiment, the medical device is an organ implant,which may in certain cases comprise autologous cells of the subject.

In a particular embodiment, the step of contacting comprises a meansselected from the group consisting of electroporation, sonication, radiofrequency, pressurized spray and combinations thereof.

In a particular embodiment, the step of contacting comprisesestablishing contact between interstitial fluid and the composition.This may be particularly advantageous for wounds which are surrounded byinterstitial fluid. Contact between interstitial fluid and thecomposition may be accomplished by piercing and/or teasing the dermiswith a needle, a microneedle, or an apparatus comprising a plurality ofneedles or microneedles. Such needles or microneedles are preferablynon-hollow and may be fashioned in a plurality for example, on a comb orbrush-like apparatus.

The method of the invention is suitable for application in humans,non-human mammals, fish and birds.

Articles of Manufacture

The method of the invention may encompass use of an article ofmanufacture which incorporates the composition comprising polymericmyrcene described herein.

The pharmaceutical composition may be in the form of a coating on thearticle of manufacture, or may be contained within a vessel which isintegral to the article of manufacture. The pharmaceutical compositionis advantageously present as a coating on devices which are inserted tothe body and are intended for integration therein, for example animplant. The pharmaceutical composition can thus promote tissue closureover the implant due to the activity of polymeric myrcene in inducingcell differentiation.

The pharmaceutical composition may be advantageously incorporated ontoor into articles used in wound healing or tissue repair, for example, adressing or bandage. The pharmaceutical composition can thus promotewound healing due to the activity of polymeric myrcene in inducing celldifferentiation.

In other cases, the pharmaceutical composition may be incorporated to adelivery device such as a needle, an injection device or a spraydispenser from which the composition is delivered to a body siterequiring therapy, for example a wound site.

Articles of manufacture include, but are not limited to a fabricarticle, a diaper, a wound dressing, a medical device, a needle, amicroneedle, an injection device and a spray dispenser. In a particularembodiment, the article of manufacture comprises a plurality ofmicroneedles. Medical devices and implants are as hereinbeforedescribed.

The following examples are presented in order to more fully illustratecertain embodiments of the invention. They should in no way, however, beconstrued as limiting the broad scope of the invention. One skilled inthe art can readily devise many variations and modifications of theprinciples disclosed herein without departing from the scope of theinvention.

EXAMPLES Example 1 Preparation of Isolated Fractions of Mastic Gum fromPlant Sources

Method 1.

Mastic resin (10 g) was combined with absolute ethanol (200 ml) and themixture was allowed to stand overnight. The mixture was shaken, largerinsoluble particles were allowed to settle over 20 minutes, and theethanol was transferred into a new flask. The remainder was shaken witha fresh portion of absolute ethanol (150 ml) for 10 minutes. Thisethanol fraction was combined with the first fraction. The procedure wasrepeated with another 150 ml portion of absolute ethanol which wascombined with first two ethanol fractions. Subsequently, the ethanol wasremoved in vacuo using a rotary evaporator (water-bath temperature 30°C.). Hexane (300 ml) was added to the remaining residue and the mixturewas shaken repeatedly over a period of two hours. After standingovernight in the closed flask in order to complete dissolution ofsoluble material and precipitation of any insoluble material, the clearhexane solution was transferred into a clean pre-weighed flask and thehexane was removed using a rotary evaporator. To the obtained isolatedfraction was added immediately the desired amount of oil and the mixturewas shaken until a homogeneous mixture was obtained.

Method 2.

Mastic resin (10 g) was combined with absolute methanol (300 ml) and themixture was allowed to stand overnight. The mixture was shaken, largerinsoluble particles were allowed to settle over 20 minutes, and themethanol soluble fraction was transferred into a new flask. Theremaining insoluble material was shaken with a fresh portion of absolutemethanol (200 ml) for 10 minutes. This second methanol soluble fractionwas combined with the first methanol soluble fraction. The procedure wasrepeated with another 200 ml portion of absolute methanol, and a thirdmethanol soluble fraction was combined with first two methanol solublefractions. Subsequently, the methanol was removed in vacuo using arotary evaporator (water-bath temperature 30° C.). Hexane (300 ml) wasadded to the remaining residue and the mixture was shaken repeatedlyover a period of two hours. After standing overnight in the closed flaskin order to complete dissolution of soluble material and precipitationof any insoluble material, the clear hexane solution was transferredinto a clean pre-weighed flask and the hexane was removed using a rotaryevaporator. To the obtained isolated fraction was added immediately thedesired amount of oil and the mixture was shaken in the closed flaskuntil a homogeneous mixture was obtained.

Method 3.

Mastic resin (5 g) was pulverized with pestle and mortar and combinedwith hexane (200 ml). The mixture was shaken every 30 minutes during aneight hour period and subsequently left to stand overnight. The hexanesoluble fraction was removed from insoluble material and transferred toa clean flask. The hexane was removed from the hexane soluble fractionusing a rotary evaporator. The remaining residue was then subjected to ahigh-vacuum system (<0.01 mbar) for at least 24 hours in order to removeadditional volatile materials. Absolute ethanol (100 ml) was then addedto the remaining residue and the mixture was shaken repeatedly over aperiod of 1 hour. The ethanol soluble fraction was transferred to cleanflask and the extraction was repeated with two additional 100 mlportions of absolute ethanol. The ethanol soluble fractions werecombined and any remaining insoluble material was allowed to settleovernight. The clear ethanol solution was transferred into a clean,pre-weighed flask and the ethanol was removed under vacuum. To theremainder was added immediately the desired quantity of oil and themixture was shaken until a homogeneous formulation was obtained.

Method 4.

Leaves, soft twigs, fruits and berries of Pistacia lentiscus L., P.atlantica or P. palestina trees were collected, cleaned and pulverized.Dissolution with ethanol or methanol was initially carried outessentially as described in Methods 1 and 2, and subsequent dissolutionswere carried out using combinations of ethanol or methanol with avegetable oil for a number of cycles.

Method 5.

Leaves (30 g) of Pistacia lentiscus L. were collected, cleaned and cutto small pieces with a knife and placed in a food processor. Olive oil(100 ml) was added and processed. The whole mixture was removed andplaced in a glass beaker. Two hundred ml of ethanol (96%) was added andthe mixture heated to 65° C. for 20 min. The whole mixture was placed ingauze and the liquid was pressed out. The upper ethanol phase wasremoved by pipetting and discarded. Residual ethanol may be removed fromthe oil phase by evaporation.

Method 6.

Berries (25 gram) of Laurus nobilis (collected in May or June) werewashed with ethanol (96%, 200 ml) for 30 seconds. The ethanol and theberries were removed and olive oil was added to the remaining residue.Any insoluble material was allowed to precipitate, and the clear oilsolution was isolated.

Method 7.

For each preparation, approximately ten grams of resin exudate collectedfrom Pistacia lentiscus L., P. atlantica or P. palestina trees in thearea of Zikhron Yaakov, Israel was used. The resin was combined with 30ml methanol in a suitable glass vessel and the mixture was vigorouslyshaken repeatedly during a time period of 30 minutes to 2 hours—. Aportion of the resin dissolved, while insoluble material settled at thebottom of the vessel. The upper liquid was decanted, and additionalaliquots of methanol were added as above, and the shaking anddecantation process was repeated. The insoluble material remaining wasthen immersed in distilled water for 30 seconds to 1 minute, resultingin a white milky liquid with insoluble material remaining. After severalalternate rapid cycles of treatment with water, and methanol, theremaining insoluble material was air dried and weighed. Typically, about1-3 grams of insoluble material were obtained from ten grams of startingresin. Similar results were obtained using ethanol as the solventinstead of methanol. Dissolution of the final fraction of insolublematerial was carried out immediately after drying by addition of avegetable oil, typically olive oil or grape seed oil, in an amountsufficient to provide a solution of desired concentration, typically 1%or 10%.

Method 8.

For each preparation, approximately ten grams of either (i) resinexudate collected from the bark of Pistacia lentiscus L. or P. palestinatrees growing in the Carmel Mountain Region, Israel, or (ii)commercially obtained Chios mastic (available for example from the ChiosGum Mastic Growers Association or from G. Baldwin & Co.) was used. Theresin was pulverized in a mortar, transferred to a glass beaker and 100ml of ethanol (98%) was added. After shaking for few minutes, theethanol was decanted, leaving a reduced mass of resin due to the removalof solubilized material. An additional amount of ethanol was added, andthe steps of shaking, decanting and solvent addition were rapidlyrepeated for a number of cycles, each cycle lasting between 5 to 30minutes. The insoluble material remaining after the final cycle(typically corresponding to 20 to 35% by weight of the commercialstarting material, or 10 to 25% of the collected resin startingmaterial) was solubilized in one of olive oil, peanut oil, grape seedoil, sesame oil, cotton oil or soy oil to give a final concentration of8 to 10% (w/w).

Method 9.

Pulverized mastic (˜10 g) was combined with 100 ml methanol. Aftershaking for few minutes, the methanol was decanted, leaving a reducedmass of non-soluble white material due to the removal of solubilizedmaterial. An additional amount of methanol was added, and the steps ofshaking, decanting and solvent addition were rapidly repeated for anumber of cycles. The insoluble material remaining after the final cycle(typically corresponding to 20 to 30% by weight of the startingmaterial) was solubilized in olive oil. The dissolution processtypically involves olive oil warmed to 45° C. and gentle agitation inthe beaker.

Method 10.

Pulverized mastic (˜10 g) was combined with 25 ml soy oil and 100 mlmethanol in a glass beaker. Stirring using a magnetic stirrer wascarried out for 2 hours. The solvent was decanted off and fresh methanolwas added, followed by stirring for one hour. The solvent was decantedoff, followed by evaporation under vacuum to remove residual solvent.

Method 11.

Pulverized mastic (˜10 g) was combined with 100 ml ethanol (96%) in aglass beaker. Stirring using a magnetic stirrer was carried out for 10minutes. The solvent was decanted off and an additional amount ofethanol was added, followed by stirring for 5 minutes and decanting offthe solvent. The steps of solvent addition, stirring and decanting wererepeated for 4 cycles. Then n-hexane (100 ml) was added to the insolublewhite material, followed by repeated shaking until the materialdissolved. A small sample was desiccated and weighed in order todetermine the concentration. The bulk of the hexane solution was appliedto a calibrated size exclusion column and the fraction having molecularweight up to 1500 was discarded. The fraction having molecular weightgreater than 1500 was mixed with 20 grams of heavy paraffin ointment.The mixture is homogenized by repeated mixing, and the hexane wasremoved by evaporation under vacuum.

This procedure may also be performed by mixing paraffins and waxeshaving increasing molecular weight in order to obtain a more solidproduct.

The term “RPh-1” is used herein to refer to an isolated fractionprepared as in any of the above Methods, and following dissolution in asuitable oil, wax or combination thereof.

RPh-1 was used directly for in vitro cell culture experiments or fortreatment of test animals, typically at final concentrations rangingfrom 0.025 to 5% in a particular oil or mixture of oils, as specifiedherein. Furthermore, as shown in Example 2, the major component of RPh-1was determined to be 1,4-poly-β-myrcene of molecular weight in the rangefrom 5000 to 20,000.

Example 2. Chemical Characterization of Polymeric Myrcene Isolated fromPlant Sources Overview

Mastic resin from Pistacia lentiscus L. was extracted according tomethod 1 or 2 in order to obtain the desired fraction (termed RPh-1)which was analyzed by Size Exclusion Chromatography (SEC) in order todefine the molecular weight distribution. The chemical structure ofRPh-1 was analyzed by nuclear magnetic resonance (NMR) followingpreparative SEC fractionation.

It was found that the RPh-1 contains a “light” fraction with molecularweights below 1,000 and a “heavy” polymer fraction with molecular weightin the range 5000 to 20,000. Based on NMR analysis (¹H-NMR and ¹³C-NMR)the predominant compound in the “heavy” fraction has a structureconsistent with that of 1,4-poly-β-myrcene.

Preparative separations were carried out using ethyl acetate andtetrahydrofuran (THF) as eluents. In both cases, the “heavy” polymerfraction was observed to exhibit various beneficial biologicalactivities, including that of inducing cell differentiation, asdescribed in Examples 4 and 6. In contrast, the “light” fractiondemonstrated toxicity in in vitro efficacy experiments using pigmentedretinal epithelial cells. It was found that in order to preserve theactivity of the polymer fraction, it is highly important to protect itfrom oxidation or cross-linking reactions by diluting it in ahydrophobic solvent, preferably oil, optionally in combination with awax.

Methods

Mastic resin (10 g) was combined with absolute ethanol (200 ml) and themixture was allowed to stand overnight. The mixture was shaken, largerinsoluble particles were allowed to settle over 20 minutes, and theethanol was transferred into a new flask. The remainder was shaken witha fresh portion of absolute ethanol (150 ml) for 10 minutes. Thisethanol fraction was combined with the first fraction. The procedure wasrepeated with another 150 ml portion of absolute ethanol which wascombined with first two ethanol fraction. Subsequently, the ethanol wasremoved in vacuo on a rotary evaporator (water bath temperature 30° C.).To the remainder was added hexane (300 ml) and the mixture was shakenrepeatedly over a period of two hours. After standing overnight in theclosed flask in order to complete precipitation of any insolublematerial, the clear hexane solution was transferred into a clean flaskand used for analytical and preparative separations.

Macromolecules are separated using Size Exclusion Chromatography (SEC)on the basis of their being excluded from the stationary phase. In SECthe highest molecular weight compounds are totally excluded from thepacking pores and therefore elute first. Molecular weights of polymertest compounds may be estimated by SEC on the basis of comparison with astandard curve constructed with compounds of known molecular weight, forexample polystyrene standards. However, polymer molecular weightsdetermined on the basis of such comparisons may be subject to aninherent error margin of at least about 10 to 15%, since therelationship between hydrodynamic volume and molecular weight is not thesame for all polymers, so only an approximate determination can be made.

For analytical SEC, a PLgel (7.5*300 mm 5μ 10³ A°) column was used andcalibrated with polystyrene standards of molecular weights 1,000, 2000,5000, 10,000, 30000 and 70000. Solvents used (hexane, ethyl acetate,tetrahydrofuran (THF), dichloromethane (DCM) and acetone) were allanalytical grade for liquid chromatography. For analytical purposes THFwas found to be optimal. The chromatography instrument used was aThermoPhinnigan TSP fitted with either a diode array detector or an ELSDdetector, using a flow rate of 1 ml/min, run time of 15 min and 100% THFfor the mobile phase.

Preparative SEC was carried out using the following conditions:

1. Conditions for THF: Column: PLgel: 25*300 mm 5μ 10³ A°

Mobile phase: hexane 60%/THF 40% flow rate 11 ml/minSeparation was repeated 12 times with 1 ml extract each and twofractions were collected:1) Heavy MW content; 2) Low MW content.

2. Conditions for DCM: Column: PLgel: 25*300 mm 5μ 10³ A°.

Mobile phase: hexane 70%/DCM 30% flow rate 11 ml/min.Separation was repeated 12 times with 1 ml extract each and twofractions were collected:1) Heavy MW content; 2) Low MW content.For each preparative SEC run, the column was calibrated with polystyrenestandards of molecular weights 1,000, 2000, 5000, 10,000, 30000 and70000.

The collected fractions from these two different mobile phases weredivided into two, one half was evaporated to dryness using an evaporatorand 3 ml oil was added. To the second half 3 ml oil was added and thenthe organic solvent was evaporated. The obtained samples were analyzedfor biological activity.

The heavy MW material from the THF elution was analyzed by ¹H-NMR and¹³C-NMR at 300 MHz and 75 MHZ respectively.

Results Analytical SEC

FIG. 1 shows the SEC analytical chromatogram obtained_using a PDAdetector (faint line) and an ELSD-SEDEX detector (bold line). A fractioncorresponding to molecular weight in the range of about 60,000 to about5000 (eluting at 5-7 minutes) was detected only with the ELSD detector.Both detectors indicated the presence of a fraction of molecular weightin the range <1,000.

Preparative SEC

FIGS. 2A-B show the heavy (FIG. 2B) and low molecular weight (FIG. 2A)fractions obtained by preparative SEC. The heavy fraction was obtainedby SEC run in DCM/hexane, while the light fraction was obtained by SECrun in THF/hexane. Table 1 summarizes the fractions obtained usingpreparative SEC and various solvent systems.

TABLE 1 Fractions collected from preparative columns using variouseluents and evaporation modes. Fraction Molecular weight No. rangeEluents/evaporation mode 19-1 Heavy THF/Hexane evaporation with oil 19-2Light 19-3 Heavy THF/Hexane evaporation without oil 19-4 Light 19-5Heavy DCM/Hexane evaporation with oil 19-6 Light 19-7 Heavy DCM/Hexaneevaporation without oil 19-8 Light

NMR Analysis

FIG. 3 shows the ¹H-NMR spectrum obtained for the heavy MW material frompreparative SEC run in hexane 60%/THF 40%. FIG. 4 shows the ¹³C-NMRspectrum obtained for the heavy MW material from preparative SEC run inhexane 50%/THF 50%.

The ¹H-NMR and ¹³C-NMR analyses indicate that 1,4-polymeric β-myrcene isthe major component of the heavy MW fraction obtained from preparativeSEC of the polar solvent-insoluble material (RPh-1) from mastic.

Example 3. Chemical Synthesis of 1,4-Polymeric β-Myrcene

Synthetic 1,4-polymeric β-myrcene preparations of various molecularweights was prepared, using methods generally based on proceduresdisclosed in Newmark et al (1988) J. Polym Sci. 26:71-77.

Methods

The following reagents were added to a 250 ml 3-necked flask equippedwith a condenser: β-myrcene, hexane and sec-butyl lithium incyclohexane, all under nitrogen atmosphere, in the quantities shown inTable 2. The volume of hexane used in each reaction was generally atleast about 20 to 25 times the volume of the butyl lithium initiator.Each reaction mixture was heated to 80° C. and stirred for about 1 hour.In order to estimate polymer concentration a small aliquot (few ml) ofsolution was taken and evaporated to dryness.

For some reaction mixtures, lithium was removed following the reactionby diluting the final mixture with an excess of hexane and washing twicewith water. The organic phase was separated and dried with sodiumsulfate.

For use in biological activity assays and characterization of molecularweight, a 10% solution of the synthesized polymer in olive oil wasprepared by adding olive oil to a final concentration of 10% (withouthexane) and the hexane solvent evaporated. Apparent molecular weight wasdetermined using SEC and calculation from a calibration curve preparedusing polystyrene standards of molecular weight 2000, 5000, 10,000,30000 and 70000. The conditions used for SEC were as follows:

Column: PLgel: 7.5*300 mm 5μ 10³ A°

Mobile phase: 100% THFFlow rate 1 ml/min

Detector: ELSD Results

The expected and calculated molecular weights of the polymeric β-myrceneproduced under different reaction conditions are presented in Table 2.

TABLE 2 Reactant quantities and product molecular weight of chemicallysynthesized polymeric myrcene. sec-butyl sec-butyl lithium lithiumβ-myrcene Expected Calculated Reaction (ml) (mol) (mol) MW MW 1 30.00420 0.0735 2381 3816.05 2 2 0.00280 0.0735 3571 7007.32 3 1 0.001400.0735 7143 11400.54 4 0.5 0.00070 0.0735 14286 27153.13 5 0.25 0.000350.0735 28571 46034.97 6 4 0.00560 0.0735 1786 2845.24

As can be seen from Table 2, the various reaction conditions yieldedpolymeric myrcene having calculated molecular weights in the range fromabout 3000 to about 46,000. The products may be designated as being inthe range of “high” molecular weight polymeric myrcene i.e. <20,000 toabout 50,000, and “low” molecular weight polymeric myrcene i.e. <3000 to˜11,000. Representative analytical SEC profiles for “high” and “low”molecular weight polymeric β-myrcene are shown in FIGS. 5A and 5B,respectively.

Reaction products washed with water yielded substantially identicalresults in analytical SEC.

FIG. 6 shows a representative ¹H-NMR spectrum of the β-myrcenepolymerization product. FIG. 7 shows a representative ¹³C-NMR spectrumof the β-myrcene polymerization product.

The ¹H-NMR and ¹³C-NMR analyses indicate that the product of thepolymerization reaction has a structure consistent with that of1,4-poly-β-myrcene.

The synthetic reaction used for producing polymeric β-myrcene involves amechanism of anionic polymerization (known as the “Michael reaction”).

For initiation to be successful, the free energy of the initiation stepmust be favorable. Therefore, it is necessary to match the monomer withthe appropriate strength of initiator so that the first addition is“downhill” A typical anionic reaction is the polymerization of styreneusing butyllithium, C₄H₉Li, in an inert solvent such as n-hexane. Whencarried out under the appropriate conditions, termination reactions donot occur in anionic polymerization. One typically adds a compound suchas water, an alcohol, molecular oxygen or carbon dioxide to terminatethe propagation, due to rapid reaction with the carbanions at the chainends.

Anionic polymerization gives rise to very sharp molecular massdistributions because transfer processes are absent. If the solvent isextremely pure, the polymer chains will still be active after all themonomer has been consumed.

The degree of polymerization is expressed as:

$n = \frac{\lbrack M\rbrack}{\lbrack I\rbrack}$

wherein M=monomer and I=initiator.

As indicated above, butyl lithium is an appropriate initiator foranionic polymerization for isoprene-containing molecules such asterpenes. Therefore, it has been used in the synthesis of1,4-polymyrcenes for the present invention.

While the above described procedure is generally disclosed in the priorart (see for example Newmark et al (1988) J. Polym Sci. 26:71-77),important modifications disclosed herein are the work up in a highdilution of hexane and the final step of changing the solvent to oil, inorder to obtain neat polymer which retains its biological activity withhigh potency.

Example 4. RPh-1 Induces Neuronal-Like Differentiation in RetinalPigment Epithelial Cell Cultures Overview

The present invention is directed to induction of differentiation andcell maturation, and has direct application to regeneration offunctional tissue, in particular neuronal tissue. Our experimentalfindings show that RPh-1 induces differentiation of retinal pigmentepithelial cells, an epithelial tissue of neuronal origin, tomorphological neuronal cells producing axons, dendrites and junctionsbetween cells known as synapses. The morphological differentiation inRPh-1 treated cells is accompanied by de novo expression of theneuron-specific differentiation antigen β3 tubulin. The induction ofneuronal cell differentiation strongly suggests that RPh-1 affectsneuronal stem cell differentiation into functional neurons. Currentdogma on the pathology of dementia and Alzheimer's disease holds thatthe deficiency involves the failure of neurons to form functionalsynaptic junctions (see for example, Kimura R, Ohno M. Impairments inremote memory stabilization precede hippocampal synaptic and cognitivefailures in 5XFAD Alzheimer mouse model. Neurobiol Dis. 2008 Nov. 5).

Accordingly, the experiments described herein support use of an isolatedfraction of mastic as described in Example 1, as well as of polymericmyrcene, an active molecule in RPh-1, as a therapeutic modality toelicit neuro-regeneration in neurodegenerative diseases such as dementiaand Alzheimer's disease.

Synthetic polymeric myrcene is also within the scope of the inventionand is useful in the therapeutic methods of the invention.

Retinal Pigment Epithelium (RPE) Cells

Studies aimed at evaluating effects of RPh-1 on various cell lines ofhuman origin led to use of ARPE-19 cells, a non-malignant human retinalpigment epithelial cell line.

The retinal pigment epithelium (RPE) is a single layer of hexagonalpigmented epithelial cells of neuronal origin, which forms the outermostcell layer of the eye retina and is attached to the underlying choroid.RPE functions include support, nourishment and protection of theunderlying photoreceptors of the neuro-retina.

RPE cells are involved in the phagocytosis of the outer segment ofphotoreceptor cells, in the vitamin A cycle where they isomerizeall-trans retinol to 11-cis retinal and in supplying the photoreceptorswith D-glucose, amino acids and ascorbic acid.

Although in vivo the RPE is pigmented, ARPE-19 cells do not form melaninand are not pigmented. In culture the cells grow as spindle shaped andas polygonal cells.

Methods

ARPE-19 cells (obtained from the American Type Culture Collection, ATCC)were plated in flat bottom 96 well tissue culture microplates (Costar)at a concentration of 2-5×10³ cells per well (1-2.5×10⁴ cells/mL) in agrowth medium consisting of DMEM:Ham F-12, 1:1, supplemented with 10%Fetal Bovine Serum, 200 mM glutamine, 100 units/mL penicillin and 100μg/mL streptomycin. The cells were allowed to adhere to the platesurfaces overnight prior to treatment with RPh-1.

RPh-1 was prepared essentially as described in Example 1, Method 1 toprovide a 10% solution in a carrier composed of grape seed oil, oliveoil, cottonseed oil, Mygliol® 810 or Mygliol® 812. The preparations wereadded to the cultures at volumes of 0.5 μl, 2 μl, 5 μl and 20 μl. Thesevolumes, introduced into an overall sample medium volume of 200 μl,correspond to final RPh-1 concentrations of 0.025%, 0.1%, 0.25% and 1%,respectively. The oil carrier served as a vehicle control and wasapplied to control cultures at the same volumes.

The cultures were incubated in a 37° C., 5% CO₂ incubator for 72 hrs.The medium was then removed, the cultures washed twice with phosphatebuffered saline (PBS), fixed with absolute methanol for 10 min andstained with Hemacolor® reagents (Boehringer Mannheim), which staincells in a manner similar to Giemsa, and may beused in a quantitativecell viability assay (see Keisari, Y. A colorimetric microtiter assayfor the quantitation of cytokine activity on adherent cells in tissueculture. J. Immunol. Methods 146, 155-161, 1992). The dye was elutedwith 20% SDS, and quantified in an ELISA reader at 630 nm (triplicatesamples evaluated). For determination of beta-3 tubulin expression,cells were plated on sterile glass coverslips immersed in 6 wellmicroplates at a concentration of 10⁵ cells/well in a medium consistingof 1:1 mixture of Dulbecco's minimal essential medium (DMEM) and Ham F12medium, supplemented with 10 fetal bovine serum and penicillin (100units/ml), streptomycin (100 μg/ml) and glutamine (2 mM). The cells wereallowed to adhere overnight to the coverslips and 7% RPh-1 in olive oil(or olive oil alone for control preparations) was administered to thecultures at a volume of 25 μl/ml medium and incubated at 37° C., 5% CO₂for 72 hrs. The cells were then washed 2× with PBS and fixed with 4%para-formaldehyde. To determine beta-3 tubulin protein expression in thecells, the glass coverslips were stained with a mouse monoclonal primaryantibody directed against human beta-3 tubulin followed by a secondaryFITC-labelled anti-mouse IgG. The cell nuclei were counter stained withDAPI. Test and control preparations were then evaluated in a confocalmicroscope.

Results

Treatment of ARPE-19 RPE cells with RPh-1 was unexpectedly found toinduce dramatic morphological changes that are unequivocallycharacteristic of neuro-differentiation. The morphological changes didnot occur in control cultures treated with oil carrier alone, andsimilar results were seen among the test cultures treated with RPh-1,regardless of the oil used as the carrier for the active compound. Themorphological changes were also associated with cessation in cellproliferation, further supporting the conclusion that RPh-1 inducesneuro-differentiation.

Control oil-treated cultures displayed the typical spindle shaped andpolygonal growth pattern characteristic of ARPE-19 RPE cells (FIG. 8A).After 48 hours of incubation in culture, treated cells treated withRPh-1 (0.1%; 1 mg/ml) were altered in shape, and developed thick,densely staining very long single protrusions reminiscent of neuronalcell axons (FIG. 8B). After 48 hour of incubation, cells treated withRPh-1 (0.25%; 2.5 mg/ml) displayed a larger number of thinner longprotrusions reminiscent of dendrites (FIG. 8C) after 72 hours ofincubation with RPh-1 the thin long protrusions formed junctions withsimilar protrusions in adjacent cells creating a network ofinter-connected cells, potentially capable of communicating informationbetween one another (FIG. 8D). Similar networks occur normally betweenneurons in the central nervous system and enable transmission andprocessing of information.

While control cells proliferated during the 72 hour incubation period,RPh-1 treated cells rapidly ceased to proliferate and the cells remainedin sparse density, further supporting the notion of celldifferentiation.

Using inactive preparations of RPh-1 which did not inducedifferentiation as described above, ARPE-19 cells began to produce largeamounts of melanin granules and these cultures continued to proliferateand cell density increased to confluence.

Treatment of ARPE-19 cells with RPh-1 (5% in cottonseed oil) was shownto result in expression of the neuronal and synaptogenesis markers β3tubulin (TUBB3), a neuronal-type differentiation marker; Arc/Arg3.1,associated with synaptic plasticity; and neuronal pentraxin II (NPTX2),a neuronal immediate early gene that functions in excitatorysynaptogenesis. Immunofluorescence analysis of differentiated ARPE-19cells showed that after 72 hours of incubation with RPh-1, the cellsstained positively for β3TUB, Arc/Arg3.1 and NPTX2 (FIG. 9, rightpanels), whereas little or no expression of these markers was seen priorto treatment (FIG. 9, left panels).

Evidence was further obtained that RPh-1 treatment of ARPE-19 cellsleads to cessation in cell replication. Cells were treated with RPh-1for 72 hours and the total protein content (related to the total numberof cells present in the culture) was compared to untreated controlARPE-19 cells. As shown in FIG. 10, the RPh-1 treated cultures containedsignificantly lower protein content as compared to control cultures,confirming that cell proliferation was substantially terminated.

A Scoring System for the Potency of RPh-1 in Inducing CellDifferentiation

On the basis of the above results, a scoring system was developed toevaluate the potency of RPh-1 for inducing differentiation in cellculture, with cells plated 2×10³ per well. The grades and theirrespective descriptions are set out in Table 3.

TABLE 3 Grade Description of Differentiation Effect 0 No effect. Thecells proliferate, the cultures become confluent and the cells maintaintheir typical spindle shaped and polygonal morphology. 1 The cellsproduce pigmented granulation, yet continue to proliferate 2 Less than10% of the cells undergo morphological changes to produce elongated,dendrite-like protrusions 3 Approximately 10-30% of the cells showelongated protrusions. Reduced cell proliferation compared to untreatedcontrol cells 4 More than 30% of cells form elongated dendrite-likeprotrusions that form junctions between adjacent cells as well as thickaxon-like extensions. 5 The entire culture undergoes differentiation.The cells remain sparse and all of them undergo morphological changesthat culminate in formation of elongated dendrite-like protrusions, axonlike structures and intercellular junctions.

Representative examples of cell cultures at grades 3, 4 and 5 arepresented in FIGS. 11A, 11B and 11C, respectively.

Example 5. RPh-1 Shortens the Recovery Period from Anesthesia

It is becoming increasingly evident that anesthesia is associated withneuronal damage, and safe effective methods are required forneuroprotection against such damage.

Methods

C57Bl/6 mice, 8 per group were injected with RPh-1 via the sub-cutaneousroute three times over 7 days (every other day) with 0.05 ml of a 3%solution in grape seed oil for a dose of 30 mg/kg. The mice were thensubjected to A sub-lethal dose (120 mg/kg) of ketamine was thenadministered to the mice. A control group was treated with 0.05 ml ofthe grape seed oil vehicle.

Results

Following anesthesia, the RPh-1 treated mice recovered significantlyfaster, as evidenced by their full mobility, while the controls werestill immobile. Recovery in the control group as defined by an abilityto become mobile took 3 minutes longer in the control group as comparedto the RPh-1-treated group. This observation indicates that the activeingredient polymeric myrcene in RPh-1 shortens the recovery period fromand can be used for neuroprotection against the adverse side effectsassociated with anaesthetic drugs.

Example 6. RPh-1 Induces Cell Differentiation Followed by Cell Death inTumor Cell Lines

The effects of RPh-1 on two melanoma cell lines and three neuroblastomacell lines were investigated. Human melanoma cell line 5151 and murinemelanoma cell line B16F10 both proliferate in tissue culture in anundifferentiated manner and do not produce melanin. Human neuroblastomacell lines Lan-1, Lan-5 and SY5Y proliferate in culture as spindleshaped cells that do not exhibit differentiation morphology.

Methods

Cells were plated at 2×10³ cells per well in 96 well flat bottommicroplates (Costar) and cultured in 200 ml of medium DMEM (Dulbecco'smedium) supplemented with 10 fetal bovine serum, 200 mM L-glutamine, 100units/ml penicillin and 100 microgram/ml streptomycin (all reagents fromGibco-BRL). Following overnight attachment, RPh-1 (from a 10% solutionin grape seed oil) was added to the cell cultures to provide finalconcentrations of 0.025%, 0.1%, 0.25% and 0.5%, and incubation wascontinued for 48 and 72 hours. The grape seed oil vehicle was used ascontrol. After 72 hours, cells were fixed with methanol and stained withHemacolor® reagents (Boehringer Mannheim).

Results

Treatment of melanoma cells with RPh-1 was found to induce formation ofmelanin after 24-48 hrs, as shown by FIG. 12B and FIG. 12C, as comparedto the control treated cells in FIG. 12A. The RPh-1 treatment furthercaused arrest of replication, as shown by the decreased cell density,for example in FIG. 12D. By 72 hours, cell death was seen in culturesincubated with each of the four RPH-1 concentrations tested.

Upon treatment of neuroblastoma cell lines Lan-1, Lan-5 and SY5Y withRPh-1 (final concentration 0.025%), the cells began to developdendrite-like protrusions and cell proliferation ceased. Higher RPh-1concentrations caused cell death in the entire culture. Thus, thetreatment with RPh-1 induced morphological neuron-like differentiationfeatures that were followed by cell death.

Conclusion

Polymeric myrcene, an active component in RPh-1, is associated with theinduction of differentiation of various cell lines derived from themalignant cancers melanoma and neuroblastoma.

A block in terminal differentiation is recognized as a major avenue inthe perpetuation of cell proliferation in cancer. Overcoming this blockhas already proven to be an effective treatment modality of severalforms of cancer (e.g. retinoids in treatment of acute promyelocyticleukemia) and is now known as “targeted therapy”. Targeted therapy doesnot kill cancerous cells but modifies their behavior, primarily byinducing differentiation. Accordingly, the aggressiveness of manycancers can be reduced.

As disclosed herein, polymeric myrcene, an active ingredient of RPh-1has been found to overcome the block in tumor cell differentiation, asindicated by formation of neuronal cell dendrites in neuroblastoma celllines, and induction of melanin formation in melanoma cell lines. Inboth cases these changes were associated with cessation in cellproliferation and cell death.

Example 7. Chemically Synthesized Polymeric Myrcene Induces CellDifferentiation in Retinal Pigment Epithelial Cell Cultures

Experiments were carried out to determine whether synthetic polymericmyrcene of two different molecular weight ranges inducesneuro-differentiation in ARPE-19 cells.

Methods

ARPE-19 cells were plated in flat bottom 96 well tissue culturemicroplates (BIOFIL) at a concentrations of 5×10³ cells per well(2.5×10⁴ cells/mL) in a growth medium consisting of DMEM:Ham F-12, 1:1,supplemented with 10% Fetal Bovine Serum, 200 mM glutamine, 100 units/mLpenicillin and 100 μg/mL streptomycin. The cells were allowed to adhereto the plate surfaces overnight prior to treatment with the chemicallysynthesized polymeric myrcene fractions.

Isolated fractions of chemically synthesized polymeric myrcene, havingdistinct molecular weights were tested for activity in the RPE celldifferentiation assay. Fraction 18-1 (molecular weight in the range ofabout 50,000 daltons), and fraction 18-2 (molecular weight in the rangeof about 20,000 daltons), described in Example 3 were used. Fractions18-1 and 18-2, and RPh-1 were each prepared at a concentration of 10% inolive oil. Each preparation was added to the ARPE-19 cell cultures usingvolumes of 0.5 μl, 2 μl, 5 μl and 20 μl, corresponding to finalconcentrations of 0.025%, 0.1%, 0.25% and 1%, respectively. Olive oilserved as vehicle control and was applied to control cultures at thesame volumes. The cultures were incubated in a 37° C., 5% CO₂ incubatorfor 72 hrs. The medium was then removed, the cultures washed twice withphosphate buffered saline (PBS), fixed with absolute methanol for 10 minand stained with Hemacolor® reagents.

Results

Both Fractions 18-1 and 18-2 were shown to have activity in inducingneuro-differentiation in ARPE-19 cells (FIG. 13 and Table 4). Optimalactivity was observed with Fraction 18-1 at 0.25% (as shown in FIG.13A), while 0.1% was somewhat effective and 0.025% had no effect (Table4). The effect of fraction 18-2 is shown in FIG. 13B.

TABLE 4 Effects of Fractions 18-1 and 18-2 on ARPE-19 celldifferentiation Fraction Volume (ul) Results 18-1 0.5 High cell density.No differentiation 2 High density. Differentiated cells 5 Lower density.Differentiated cells. Long axons with intercellular junctions 20 Celldeath 18-2 0.5 Low density. Few full differentiated cells. 2Differentiated cells but axons shorter and less prevalent than 18-1 5-20Cell death RPh-1 0.5 Differentiated cells in clusters. Long axons 2Differentiated cells with lower density. Long axons 5-20 Cell death OilVehicle 0.5-20   Very high cell density, no differentiation

Conclusion

The observed results support the conclusion that RPh-1, a formulation ofan isolated fraction of mastic gum, has activity in inducingdifferentiation of neuronal cells.

The observed results also support the conclusion that polymeric myrcene,whether isolated from a plant source or that chemically synthesized, hasactivity in inducing differentiation of neuronal cells.

Example 8. The Effect of RPh-1 in Inducing Cell Differentiation isBlocked by the Polar Solvent-Soluble Fraction Present in Mastic ResinOverview

Mastic resin and various compounds identified therein have beenassociated with a variety of beneficial biological and therapeuticactivities. Various prior art disclosures indicate that the biologicalactivity is associated with a fraction that is obtained by extraction ofmastic with a polar solvent, and recovery of the polar solvent-solublematerial. In contrast, RPh-1 is a fraction which has been isolated frommastic resin on the basis of its being soluble in both polar organicsolvents and non-polar organic solvents, while compounds that aresoluble only in polar organic solvents but not in non-polar organicsolvents are discarded (the latter herein designated Fraction SP). Amajor component in RPh-1 is polymeric myrcene, as shown in Example 2.This compound however, has not previously been attributed withbeneficial effects, but rather has been acknowledged to interfere withoral administration and bioavailability of active compounds present inmastic resin. Fraction SP corresponds to prior art mastic fractionswhich have been attributed to have various beneficial biologicalactivities. The aim of the present study was to assess the effect of SPon the cell differentiation effect exerted by RPh-1. It is now disclosedthat compounds present in SP interfere with and block the celldifferentiation effects induced by RPh-1.

Methods

Mastic resin was treated to obtain RPh-1, essentially as described inMethod 1 of Example 1, using ethanol as the polar solvent. Theethanol-soluble fraction was decanted off from the insoluble material toobtain Fraction SP. Mixtures of RPh-1 and Fraction SP in differingproportions were prepared as follows:

Mixture RPh-1 (%) Fraction SP (%) A0 95 5 A1 90 10 A2 80 20 A3 70 30 A450 50 A5 25 75

In addition, whole mastic dissolved in oil (warmed to 60° C.) wasprepared to obtain preparation TC.

The results of the study, summarized in Table 5, indicate that fractionsrich in RPh-1 (A0 and A1) were effective in inducing ARPE-19differentiation. The morphological changes seen in these cultures weresimilar to that shown in FIGS. 8B and 8C. As the proportion of FractionSP was increased in the mixtures, cell death increased, with no celldifferentiation observed. Cells in cultures treated with SP alone weredead at all tested doses, and fraction TC exerted only negligibleeffect.

These results show that the potent neuro-differentiation inducingactivities were only contributed by the polymers in RPh-1 whereas the SPpolar fraction only caused cell death.

TABLE 5 Effects of mixtures of RPh-1 and SP on cell differentiationFraction Volume (ul) Results A0 0.5 High cell density, differentiatedcells. 2 Lower cell density. Differentiated cells with long axons.  5-20 Cell death. A1 0.5 High cell density. Less differentiated cellsthan in A0. 2 Differentiation. 5 Low cell density. Differentiated cellswith long axons 20 Cell death. A2 0.5 Low cell density. Differentiation.2 Partially differentiated cells (only short dendrites) associated withcell death 20 Cell death. A3 0.5 Sporadic, partial differentiation. Highcell density (cell proliferation).   2-20 Cell death. A4 0.5 Cell death,toxic   2-20 Cell death. A5 0.5-20 Cell death. RPh-1 2 Differentiatedcells with intercellular junctions and long axons 5 Differentiation andlong axons 20 Cell death. SP 0.5-20 Cell death. TC 0.5 Negligibleeffects 2 Cell death. Vehicle 0.5-20 High density

Example 9. Wound Healing in Dogs

An aging Golden Retriever male dog had an open chronic leg wound formore than 6 months. The dermal lesion was associated with alopecia (lossof fur) and depigmentation of the surrounding fur. The dog was treatedby several cycles of topical treatment with RPh-1. Following the initialapplication, transient edema with swelling occurred for 16-20 hrs. Thiswas followed by de novo formation of functional epithelial tissue(epithelization) and neoangiogenesis (novel formation ofmicrovasculature) with normal tissue contours, resulting from rapid andvigorous formations of granulation tissue. Wound healing contractedinwards towards the center of the wound, suggesting the presence offibro-myocytes (of mesodermal origin).

The wound was completely healed within approximately 12 weeks withpredominantly functional skin and re-growth of the fur. FIG. 14 showsthe afflicted area before (FIG. 14A) and after (FIG. 14B) treatment withRPh-1.

In another aging male dog afflicted by alopecia, topical treatment withRPh-1 resulted in re-growth of the fur to become integrated with thesurrounding fur.

A different dog had a jaw tumor (non-induced), portions of whichprotruded into the buccal cavity. The protruding portions weresurgically excised, while the sections of the tumor that were embeddedwithin the jaw could not be removed. The tumor was diagnosed as asarcoma. RPh-1 formulated in grape seed oil was applied to the affectedjaw area. The treatment brought about complete cure of the gums coveringthe surgical incision site to the extent that no scar was left and thesurgical incision site was no longer discernable. Even the expectedrecurrence of the tumor from portions embedded in the jaw was preventedfor an extended interval of several weeks. The treatment with RPh-1induced an extraordinarily rapid healing of the surgical incision siteand complete regeneration of the gums.

In both of the above cases, wound healing was accompanied by a generalincrease in vitality, mental awareness and physical activity in thetreated dogs.

The above results support the use of polymeric myrcene, the activecomponent of RPh-1, for wound healing, regeneration of hair folliclesand reversal of neurological degeneration.

Example 10. Treatment of Wounds in Fish

Gold fish as well as koi fish (both in the carp family) are prone tointegument ulcers caused by bacteria, in particular Aeuromonashydrophila.

Gold fish weighing approximately 100 gram each, which had developedbacterial ulcerations were divided into two groups in separate tanks,each group containing four fish. Each tank was filled with a volume of100 liters of water and maintained under aeration with an air pump. Thegroups were randomized by weight and wound size (in the range of 1-1.5cm by 1-1.5 cm). Each fish was injected intramuscularly through intactintegument at a site approximately 5 mm from an ulcer with 20microliters of either grape seed oil alone (control group), or a 1%solution of RPh-1 in grape seed oil (treatment group).

Fish in the test group began to improve progressively following 4 cyclesof treatment with RPh-1 and were healed over a period of a month. Allfish in this group survived through the six week duration of the study.These fish also exhibited alert and responsive behavior including activeswimming, searching for and snatching at food provided at the watersurface, and rapid, startled movement away in response to percussion onthe wall of the tank.

In contrast, fish in the control group displayed no improvement in thecondition of their ulcers. The fish were lethargic, exhibited sedentarybehavior at the bottom of the tank, and did not respond to stimulation.All of the fish in this group died by the end of six weeks.

The differences between these two groups were highly significant in bothparameters: fish survival and wound closure.

Example 11. Effect of RPh-1 in Wound Healing Using B6.V-Lepob/OlahsdMice Model

B6.V-Lepob/OlaHsd (ob/ob) mice (express obesity at age 4 weeks) wereused to evaluate the effect of RPh-1 in wound healing. Full thicknessskin puncture was performed using a disposable biopsy puncher(Uni-Punch® Disposable Biopsy Punch, Premier) in the distal zone of eachmouse back. The puncture has an ellipse shape. Average long axis lengthof punctures ranged between 5.1 to 5.3 mm. The average width axis lengthof punctures ranged between 4.8 to 5.1 mm RPh-1 (5%) in olive oil wasinjected subcutaneously at two sites surrounding the wound at a distanceof 3-5 millimetres from the edge of the wound (Group A, n=6) ortopically onto the wound (Group B, n=6). Vehicle was applied topicallyonto the wounds of mice (Group C, n=6). Thereafter, RPh1 (5%) wasapplied 3 times a week, 7 times in total, during the 16 days of thestudy at a 20 ul dose volume (injection) or a 50 ul dose volume (topicaladministration).

FIG. 15 shows that at day 11 following wound infliction, the size of thewound (wound area) was significantly reduced in mice treated with RPh-1(Group A) as compared to those treated with vehicle alone (p=0.005)(Group C). The rate of wound healing during the period from Day 0 to Day11 following wound infliction was significantly more rapid in micetreated with RPh-1 as compared to those treated with vehicle alone(p-0.034).

Example 12. Effect of RPh-1 in Reversing the Neurodegenerative Effectsof Chronic Cerebral Hypoperfusion (Vascular Dementia) in a Rat Model

Vascular dementia (VD) is a subtype of dementia with a prevalence thatis second only to that of Alzheimer's disease in westernized societies.VD causes many neuropsychiatric and physical problems, and represents asignificant economic burden. Brain imaging has revealed obvious changesin the cerebral cortex and white matter, and these lesions are thoughtto be the core pathology for cognitive declines in patients withvascular dementia (see for example, Farkas et al., Experimental cerebralhypoperfusion induces white matter injury and microglial activation inthe rat brain. Acta Neuropathol. 2004; 108:57-64; Stenset et al., Whitematter lesion subtypes and cognitive deficits in patients with memoryimpairment. Dement Geriatr Cogn Disord. 2008 26: 424-431).

Cerebral lesions can be experimentally induced in rat brains bypermanent occlusion of both common carotid arteries which can affectcognitive function. This model is similar to vascular dementia and theexperimental technique can decrease the blood flow in the cerebralcortex and hippocampus by up to 40-80% for several months, inducingcertain learning disorders. Thus this model was used to study theeffects of RPh-1 treatment in reversing the deficiencies caused byvascular dementia lesions.

A total of 40 animals were randomized into 3 groups i.e. an untreatedsham control group, a vehicle control group and an RPh-1 treated group(10-15 animals per group). They were randomized into 3 groups, anuntreated sham control group, a vehicle control and an RPh-1 treatedgroup. Ten μl of RPh-1 (5% in cottonseed oil) or vehicle wasadministered subcutaneously 2×/wk, with the first dose administered 14days after induction of vascular dementia.

The Morris water maze (MWM) test is sensitive to hippocampal function.The water maze task is performed to evaluate two CCA-related learningdeficits using the method described previously (Watanabe et al.,Cilostazol Stroke. 2006; 37(6):1539-1545). In a 160-cm diameter circularpool filled with 20-cm deep water, a circular transparent acrylicplatform is prepared, the top surface of which is 3 cm below the water.Rats are released facing the wall, and the time taken to escape to theplatform is recorded as the escape latency. Tests are performed on day 3before CCA occlusion and on days 14, 35, 56, 84 and 112 after CCAocclusion. On training days six training trials are conducted per daywith an inter-trial interval of 2 min. Animals are placed in the pool atone of six starting positions. In each training trial, the time and pathlength required to escape onto the hidden platform are recorded. Resultsof six training trials are averaged to obtain a single representativevalue, and the averages are used for final statistical analyses. Animalsthat found the platform are allowed to remain on the platform for 30sec. Animals that do not find the platform within 90 sec are softlyguided to the platform for 30 sec at the end of the trial.

Performance of RPh-1-treated animals (cross-hatched bars), vehicletreated animals (open bars) and in sham control animals (black bars)were tested for frequency in platform location (FIG. 16A); the timespent in platform area (FIG. 16B); the latency to find the platform(FIG. 16C); the frequency in zone 1 location (FIG. 16D); the time spentin light part (FIG. 16E); the latency to find the platform (FIG. 16F);and the velocity (FIG. 16G). All tests showed significantly higherperformance in the RPh-1-treated animals as compared to at least one ofthe control groups.

Example 13. Pathologic Weight Control Regulation Effect of RPh-1(Orexigenic and Anti Obesity Effect)

The dogs with various wounds described in Example 9 additionallysuffered from loss of appetite and would not eat food placed in front ofthem. Following after approximately 10 days of treatment with RPh-1 asdescribed, dogs gradually re-gained interest in food and started to eat.Within a month, the dogs showed strong interest in food and appetite wassimilar to that of normal healthy dogs.

The fish with ulcerations described in Example 10 additionally sufferedfrom loss of appetite. The control group continued to ignore foodapplied into the water, whereas the fish treated with RPh-1 respondedeagerly with rapid movement in response to administration of food.

Rats described in Example 12 additionally suffered from weight lossafter chronic cerebral hypoperfusion. After 35 days of treatment (day 56of study) rats treated with RPh-1 as described, recovered their weightsignificantly faster than animals treated with vehicle (FIG. 17A).

Mice described in Example 11 generally suffer from obesity as a resultof mutation leptin gene. FIG. 17B shows that subcutaneous administrationof RPh-1 to mice (Group A; diamond symbols), causes a significant lowerbody weight gain compared to vehicle treated animals (Group C; trianglesymbols) or animals treated by topical administration of RPh-1 (Group B;square symbols). Mice of group A gained 4.9% during the 11 days. Thebody weight gain was compared of the initial (day 0) body weight. Thebody weight gain of group A is significantly lower than the mean bodyweight gain of mice in group B (p value=0.02, T-TEST, Excel). Mice ofgroup C were similar (p=0.08) to mice of group B and gained body weightsignificantly different (p value=0.04) from mice of group A. Mice ofgroup B and C gained 10.2% and 9.1% respectively. The rate of bodyweight gain in all groups as expressed by the slopes was similar (p=0.07(A vs. B), 0.08 (A vs. C) and 0.43 (B vs. C).

The above observations support the conclusion that RPh-1 is regulator ofpathological weight disorder and can serve as an orexigenic (appetitestimulant) or anti-obesity agent.

Example 14. Effect of RPh-1 in Transient Middle Cerebral ArteryOcclusion (tMCAO) Stroke Model in Rats

In a study to assess the ability of RPh-1 to prevent or reverseneurological deficit as a result of ischemia utilizing the rat transientmiddle cerebral arterial occlusion model (tMCAO), RPh-1 (5% incottonseed oil) was administrated subcutaneously at a 10 ul dose andfirst administration was done 3 h after the surgical procedure and thentwice weekly until termination of the study on day 28. During the studyneurological, motor and somatosensory functions were tested in a batteryof behavioral tests.

Throughout the study no significant differences in general physiologicalconditions, body weight gain or general clinical signs between the twogroups were observed.

Clear differences were seen between the RPh-1 treated group and thevehicle treated control group in neurological function recovery afterstroke during the 28 days following stroke. In general, accelerated andimproved recovery was demonstrated in animals that were treated withRPh-1. Somatosensory functions were most sensitive to the treatment, andsignificant response was demonstrated as early as day 8 following stroke(FIGS. 18A and 18C). Assessment of Neuroscore showed significantdifferences were seen only in rats treated with RPh-1 (Group A), betweenday 8 and day 14, and between day 8 and day 28 (FIG. 18A). Neurologicalrecovery as assessed by the patch removal test was significant only inrats treated with RPh-1 (Group A) between day 2 and the other days (FIG.18C). Motor function improvement, as assessed by the stepping test, wassignificant only in rats treated with RPh-1 (black bars), by day 28(FIG. 18B).

Example 15. Effect of RPh-1 on Retinal Ganglion Cells (RGC)

Axotomy of the optic nerve was performed on the right eye of deeplyanesthetized rats (19 rats per group). The test group received asub-dermal injection in the posterior neck area of RPh-1 (5% incottonseed oil); 0.025 ml/injection), and the control group wassimilarly injected with the same volume of vehicle. The first injectionwas given to all the animals directly after surgery. Subsequentinjections (same dosage and method of administration) were administeredtwice a week, every 3 to 4 days.

Fourteen days after axotomy, a fluorescent retrograde neurotracer(Di-Asp) was inserted into the axotomized optic nerve in order to stainsurviving Retinal Ganglion Cells (RGC), and 24 hours later, the ratswere sacrificed in a CO₂ saturated chamber and the injured right eye wasenucleated. The retinas were isolated, flattened on a slide and fixedwith xylene based mounting medium.

Whole-mount retinas were evaluated with a fluorescent microscope. Dyedcells were counted manually. The average number of RGC per group isshown in FIG. 19, showing a significantly higher number of cells in theRPh-1 test group.

Example 16. Retinal Detachment (RD) Model

Retinal detachment (RD) was performed on the right eye of deeplyanesthetized animals (xylazine 50 mg/kg and ketamine 35 mg/kg) followingdilatation of the pupil with Tropicamide drops 0.5%. RD was inducedthrough the generation of a small opening in the retina at the oraserata followed by a sub-retinal injection of 5 μl saline with a 30 Gsyringe needle. Approximately half of the retinal area was detached bythis procedure.

Rats with RD were divided into two experimental groups, with the testgroup receiving a sub-dermal injection in the posterior neck area ofRPh-1 (5% in cottonseed oil; 0.025 ml/injection), and the control groupinjected with the same volume of vehicle. The first injection was givento all the animals directly after surgery. The second injection (samedosage and method of administration) was administered 48 hours aftersurgery.

On days 3 and 14 days after RD, the operated rats were euthanized in aCO₂ saturated chamber. The injured right eye and the untreated left eyewere enucleated. The retinas were isolated, frozen on dry ice andprocessed for Western blot analysis or immunohistochemical analysis. Theleft eye retinas served as non-operated controls.

The expression levels of Semaphorin3A (Sema3A), Neuropilinl (NP1), andGAP43 were studied, Caspase3 was used as a apoptotic marker, andmorphological changes in Müller and microglial cells were examined.

Sema3A is an axonal growth inhibitor that has been shown to be involvedin retinal ganglion cell loss following injury to the optic nerve. Highlevels of Sema3A were detected in retinas after RD as shown by Westernblot analysis (FIG. 20A). Treatment with RPh-1 clearly decreased Sema3Aexpression levels, both in control non-injured retinas and those with RD(FIG. 20A). Samples were normalized to beta-actin expression (lowerband, FIG. 20A).

Immunohistochemical analysis of 20 μm retinal sections incubated withanti-Sema3A antibody and the nuclear dye Sytox Blue showed that Sema3Aexpression was clearly higher in detached retinas as compared to thecontrols. Sema3A expression was observed mainly around the retinalganglion cells. Similar to the results observed in Western blotanalysis, Sema3A expression was reduced in RD animals treated withRPh-1.

NP1 is a functional Sema3A receptor. TUNEL-positive cells, indicatingapoptotic processes, were evident 24 hours post retinal detachment andincreased after 7 days.

Caspase-3 was activated in response to RD. However, caspase-3 waselevation was significantly attenuated in RD animals treated with RPh-1(FIG. 20B).

GAP43 is an intracellular protein that is tightly connected to themembrane of the growth cones. It is normally expressed during theprocess of synaptogenesis. In the retina, GAP43 is expressed in theneurons at an early stage of embryogenesis, while the optic nerve isstill elongating. In the rat optic nerve, GAP43 is found both in axonsand cell bodies of RGCs, but the expression disappears at the age of 8to 16 weeks, and is found again following ischemia or injury to theoptic nerve.

The morphological changes of the Muller cells were studied by stainingfor glial fibrillary acidic protein (GFAP). GFAP labels Muller cells inthe retina, and is commonly used as a stress indicator. GFAP labeling inthe intact control retina was concentrated at the GCL.Immunohistochemical analysis showed elevated levels of GFAP in thedetached retinas in comparison to controls. Detached retinas treatedwith RPh-1 exhibited higher GFAP levels.

Microglial invasion and activation are regarded as harmful or beneficialto neurons. Microglial activation after acute CNS injury is primarily areactive and adaptive glial cell response, which is triggered by injuredneurons and which is designed to ameliorate primary tissue damage and topromote subsequent repair and gliosis (glial scar) as a result.Microglia become activated in the retina usually after injury stimulateand recruit endothelial cells and fibroblasts. Immunohistochemicalanalysis of sections of detached and non-injured retinas labeled withIB4 and stained with the nuclear dye PI showed evidence of activatedmicroglial cells in detached retinas only. However, in detached retinasfrom animals treated with RPh-1, less microglial activation was evidentas compared to detached retinas from animals that were treated withvehicle.

The results showed reduced recruitment of active microglia around aninjury region and support a scar-less repair mechanism of wounds.

Example 17. Preparation of Complexes of Cyclodextrin with PolymericMyrcene

Cyclodextrins, by virtue of their ability to form inclusion complexeswith many drugs, can substantially increase the aqueous solubility ofbiopharmaceuticals, in particular those that are defined aswater-insoluble such as polymeric myrcene. Cyclodextrins arewater-soluble compounds, which can form reversible complexes with poorlywater-soluble molecules resulting in a soluble molecular inclusioncomplex. When the inclusion complex of the drug-cyclodextrin combinationis diluted in a sufficiently large volume of water or blood, itdissociates rapidly, releasing the sequestered pharmacologically activeagent.

Complexation of polymeric myrcene with β-HPCD will be performed asfollows:

a. Dissolution of pre weighed polymeric myrcene in a minimum amount ofnon-polar solvent such as hexane, heptane, or the like.b. Dropwise addition of the non-polar solvent to the β-HPCD powder.c. Drying at 50-80° C. until non-polar solvent evaporates.d. Mixing with necessary amount of water.e. Dissolution with sonication and heating.f. Filtration through 0.2-0.45 μm filter.

Example 18. Preparation of Nanoemulsions of Polymeric Myrcene

Liquid oil-in-water nanoemulsion formulations are to be prepared by highpressure emulsification techniques of all lipid ingredients and theactive component polymeric myrcene dissolved in the lipid oil phase andemulsified with an aqueous phase, projected to result in the formationof stable, spheric and uniformly dispersed drug-containing lipidnanodroplets. The emulsion droplet size reduction is essential togenerate drug formulations with high stability. Preferred nanoemulsiondroplets have a mean droplet size of less than one micron (generally inthe range of 0.1-0.2 μm) uniformly dispersed in an aqueous phase. Theuniqueness of the large internal hydrophobic oil core of thenanoemulsion droplets provides high solubilization capacity for waterinsoluble compounds such as polymeric myrcenes.

1. Preparation of Oil Phase

The oil phase is composed of 13% lipoid E-75, 0.026% αTP-succinate,propylparaben as antioxidant and 86.9% Miglyol® 810. Polymeric myrceneprepared as in Example 1 is dissolved in the oil phase. The componentsare mixed with mild heating until a homogenous completely solubilizedsolution is obtained.

2. Preparation of Aqueous Phase

The aqueous phase is composed of 0.1% EDTA, 0.5% Tween-80, 2.3%glycerol, methylparaben as preservative and 97.1% water. pH was adjustedto 7.4 by NaOH 1N.

3. Mixing of Oil and Aqueous Phases

Oil phase (3.7 g) is heated and added to 70 ml of the aqueous phase(preheated). The mixture is gently stirred for 10-15 min at roomtemperature.

4. Preparation of Oil-in-Water Coarse Emulsion

An oil-in-water emulsion is prepared using the medium size dispenser andhigh shear homogenizing unit Polytron®, at 20,000 rpm for 5 min.

5. Sizing the Emulsion to Submicron Range by Gaulin® High PressureHomogenizer

The droplet size of the emulsion obtained after step 4 is reduced to thesubmicron (nanosize) range by submitting the emulsion to high shearhomogenization using the Gaulin® Microlab 70 high pressure homogenizerat 800 bar pressure. A total of 5-6 cycles should be performed to obtainhomogenous nanoemulsion droplets having average particle size of lessthan 200 nm. Particle size is to be determined by photon correlationspectroscopy (PCS) using a N4MD particle size analyzer (Coulter®Electronics, UK). When most of the particles (>90%) are smaller than 200nm, the sizing process is determined to be complete.

6. Sterile Filtration

Filtration at aseptic conditions of the nanoemulsion to sterile vialsusing a 0.2 μm PES sterile filter and storage at 40° C.

Example 19. Preparation of Spray-Dried Polymeric Myrcene Powder

A convenient process for manufacturing the polymeric myrcene-lipidmixture product is by direct spray-drying of the formulation from amixture of non-polar solvent dispersion containing all the lipidingredients and water containing the hydrophilic components, taking intoaccount cost effectiveness and upscaling considerations. The selectedspray-drying method is optimized in order to get a fine, free-flowingpowder. Polymeric myrcene is dissolved in the lipid phase containing thelipid ingredients lecithin, tricaprin (capric acid triglyceride),tocopherol succinate and warmed (˜40° C.) in a nonpolar solvent until agood dispersion is obtained. A dispersion of fumed silicon dioxide(Cab-O-Sil®) in water (5%) was prepared by swelling the powder inpurified water. The resultant slurry (prewarmed to 40° C.) is thenpoured slowly into the nonpolar solvent lipid dispersion and the mixtureis agitated at 40° C. for about 1 hr until a homogenous dispersion isobtained. The mixture is then spray-dried using the Yamato Pulvis® GA32spray-dryer. The spray-drying conditions are: flow rate 7 ml/min, inlettemperature 130° C., outlet temperature 70° C., and drying air flow 0.5m³/min. A homogeneous dry powder containing the polymeric myrcene-lipidmixture is expected to be obtained.

The polymeric myrcene-lipid mixture formulation prepared by the directspray drying process is expected to show good water dispersibility, thusbeing suitable for the preparation of solid-dosage forms such as hardgelatin capsules or tablets for the enhanced oral delivery of polymericmyrcene with potential good oral bioavailability.

Example 20. Preparation of Liposomal Preparations Containing PolymericMyrcene

Lipids containing dissolved polymeric myrcenes were dissolved in 100 mldichloromethane in a round bottom flask, and stirred for 30 min at roomtemperature until a clear transparent solution was obtained. Solventwill be evaporated using a rotary evaporation unit at 39° C. First, theflask will be rotated at 4.5 rpm, 5 min under atmospheric pressure,followed by 10-30 min (until full evaporation of the solvent) under weakvacuum, and finally 15 min under full vacuum. At the end of theevaporation process a uniform lipid film will be created. The lipid filmwill be dissolved in 15 ml isotonic buffer. Liposomes are prepared byvigorous shaking for 10-30 min using multi-wrist shaker, until a uniformand milky dispersion of multilamellar vehicle (MLV) will be formed andno remaining lipid film will be apparent. In order to obtain anequilibrated and homogenous liposome preparation the flask will befurther shaken at 37° C. for 30-90 min. at 270 rpm.

Example 21. Preparation of Microemulsions Containing Polymeric Myrcenes

Several surfactants commonly used in parenterals may be utilized todevelop water-in-oil and oil-in-water-microemulsions acceptable forinjectable, oral and topical use. The pharmaceutically acceptablesurfactants suitable for the formation of microemulsion formulations arenon-ionic surfactants including polyoxyl 40 hydrogenated castor oil(sold under the trade name Cremophor RH40®), polyoxyl 35 castor oil(sold under the trade name Cremophor® EL), polyoxyethylene sorbitanfatty acid esters (polysorbates), poloxamers (Pluronics®), vitaminE-TPGS 1,000 (VE-TPGS 1,000), polyoxyethylene alkyl ethers, Solutol®HS-15, Tagat® TO, Peglicol 6-oleate, polyoxyethylene sterates, orsaturated polyglycolyzed glycerides, all of which are commerciallyavailable. The preferred surfactants include polyoxyl 40 hydrogenatedcastor oil (Cremophor® RH40®), polyoxyl 35 hydrogenated castor oil(Cremophor® EL), polyoxyethylene sorbitan fatty acid esters(polysorbates), poloxamers (Pluronics®), and vitamin E-TPGS 1,000. Thetotal amount of the surfactant present in the composition will begenerally from about 100 to about 700 mg/g, and preferably from about300 to about 500 mg/g.

Preparation of microemulsions containing polymeric myrcene may beperformed by dissolving the polymeric myrcenes in an appropriate amountof oil such as medium chain tryglycerides (Miglyol) in a suitable vial.The vial is then capped. The vial is put into a water bath of about50-60° C. and shaken gently until all of the drug material is completelydissolved. After the vial is cooled to room temperature, an appropriateamount of surfactant (such as Cremophor® EL or VE-TPGS) is added andfollowed by the mixture of mono- and di-glycerides of fatty acids, ifany. The vial is then capped and placed into the water bath of about50-60° C. The vial is shaken gently to obtain a clear, uniform solution.This solution can be filled into HPMC capsules and stored at roomtemperature before oral dosing. Alternatively, the substituted polymerpowders (such as HPMC) can be added into the solution with adequateagitation (i.e., stirring, shaking) to obtain a uniform polymersuspension. The resulting composition can then be filled into eithersoft gelatin or hard gelatin capsules and stored at room temperaturebefore oral dosing. Alternatively the microemulsion formulation can beused as a topically or filtered through 0.2 um membranes to beadministered parenterally.

The microemulsions containing polymeric myrcenes have goodwater-dispersibility properties and self-emulsify when diluted inaqueous media to form small nanometric micelles that with enhancedbioavailability.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without undue experimentation and withoutdeparting from the generic concept, and, therefore, such adaptations andmodifications should and are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. It is tobe understood that the phraseology or terminology employed herein is forthe purpose of description and not of limitation. The means, materials,and steps for carrying out various disclosed functions may take avariety of alternative forms without departing from the invention.

1. A composition consisting of an isolated fraction of mastic gum and apharmaceutically acceptable carrier, wherein the fraction ischaracterized in that it is soluble in at least one polar organicsolvent and in at least one non-polar organic solvent, and wherein saidfraction is substantially devoid of compounds which are soluble in saidpolar organic solvent but insoluble in said non-polar organic solvent,wherein the fraction is obtained by a process comprising the steps of:(a) treating mastic gum with a polar organic solvent; (b) isolating afraction soluble in said polar organic solvent; (c) treating the solublefraction obtained in step (b) with a non-polar organic solvent; and (d)isolating a fraction soluble in said non-polar organic solvent.
 2. Thecomposition according to claim 1, wherein the mastic gum is obtainedfrom a species of Pistacia selected from the group consisting of P.lentiscus, P. atlantica, P. palestina, P. saportae, P. terebinthus, P.vera and P. integerrima.
 3. The composition according to claim 1,wherein the at least one polar organic solvent is selected from thegroup consisting of an alcohol, an ether, an ester, an amide, analdehyde, a ketone, a nitrile, and combinations thereof; or wherein thepolar organic solvent is selected from the group consisting of ethanol,methanol, propanol, isopropanol, 1-butanol, 2-butanol, sec-butanol,t-butanol, 1-pentanol, 2-pentanol, 3-pentanol, neopentanol,3-methyl-1-butanol, 2-methyl-1-butanol, 3-methyl-2-butanol,2-methyl-2-butanol, ethyleneglycol, ethyleneglycol monomethyl ether,diethyl ether, methylethyl ether, ethylpropyl ether, methylpropyl ether,1,2-dimethoxyethane, tetrahydrofuran, dihydrofuran, furan, pyran,dihydropyran, tetrahydropyran, methyl acetate, ethyl acetate, propylacetate, acetaldehyde, methylformate, ethylformate, ethyl propionate,methyl propionate, dichloromethane, chloroform, dimethylformamide,acetamide, dimethylacetamide, N-methylpyrrolidone, acetone, ethylmethylketone, diethyl ketone, acetonitrile, propionitrile, and combinationsthereof; or wherein the at least one non-polar organic solvent isselected from the group consisting of acyclic or cyclic, saturated orunsaturated aliphatic hydrocarbons and aromatic hydrocarbons, each ofwhich is optionally substituted by one or more halogens, andcombinations thereof; or wherein the non-polar organic solvent isselected from the group consisting of C5-C10 alkanes, C5-C10cycloalkanes, C6-C14 aromatic hydrocarbons and C7-C14 perfluoroalkanes,and combinations thereof; or wherein the non-polar organic solvent isselected from the group consisting of pentanes, hexanes, heptanes,octanes, nonanes, decanes, cyclopentane, cyclohexane, cycloheptane,benzene, toluene, xylene, and isomers and mixtures thereof; or whereinthe non-polar organic solvent is hexane.
 4. The composition according toclaim 1, wherein said process further comprises the steps of: (e)optionally removing, prior to step (c) the polar organic solvent of step(b); and/or (f) optionally removing, after step (d) the non-polarorganic solvent.
 5. The composition according to claim 4, wherein saidprocess further comprises the step of size fractionating the fractionobtained in step (b) or step (d); or wherein either or both of steps (e)and (f) comprise removing the solvent by a means selected from the groupconsisting of rotary evaporation, application of high vacuum and acombination thereof; or wherein said process further comprises repeatingsteps (a) to (b) and/or steps (c) to (d) for a multiplicity of cycles.6. The composition according to claim 1, wherein the fraction issubstantially devoid of terpene compounds which are soluble in saidpolar organic solvent and insoluble in said non-polar organic solvent.7. The composition according to claim 1, wherein the fraction containspolymeric myrcene having a number average molecular weight in the rangeof at least about 1,000 to about 20,000.
 8. The composition according toclaim 7, wherein the polymeric myrcene is selected from the groupconsisting of polymeric β-myrcene (poly-β-myrcene), polymeric α-myrcene(poly-α-myrcene), myrcene copolymers and combinations thereof; whereinthe poly-β-myrcene is selected from the group consisting ofcis-1,4-poly-β-myrcene, trans-1,4-poly-β-myrcene, 3,4-poly-β-myrcene,1,2-poly-β-myrcene and combinations thereof.
 9. The compositionaccording to claim 1, wherein the isolated fraction of mastic gum isfrom about 0.01 to about 25% (w/w) of the total weight of thecomposition.
 10. The composition according to claim 1, wherein thecarrier is a hydrophobic carrier which is selected from the groupconsisting of: a) at least one oil selected from the group consisting ofcottonseed oil, almond oil, canola oil, coconut oil, corn oil, grapeseed oil, olive oil peanut oil, saffron oil, sesame oil, soybean oil andcombinations thereof; b) at least one wax; and c) combinations thereof.11. The composition according to claim 1, which is in a form suitablefor cosmetic or dermatologic administration; or in a form suitable foradministration by a route selected from the group consisting of oral,topical, parenteral, intramuscular, subcutaneous, intradermal, vaginal,rectal, intracranial, intranasal, intraocular, auricular, pulmonaryintralesional, intraperitoneal, intraarterial, intracerebral,intracerebroventricular, intraosseus and intrathecal; or in a formselected from the group consisting of a capsule, a tablet, asuppository, a suspension, an ointment, a cream, a lotion, a solution,an emulsion, a film, a cement, a powder, a glue, an aerosol and a spray.12. The composition according to claim 1, which is: a) an inclusioncomplex and the carrier contains at least one cyclodextrin; b) ananoemulsion in which droplets have an average particle size of lessthan 800 nm; or c) a microemulsion and the carrier contains at least onenon-ionic surfactant selected from the group consisting of a polyoxylcastor oil, a polyoxyethylene sorbitan fatty acid ester (polysorbates),a poloxamer, a vitamin E derivative, a polyoxyethylene alkyl ether, apolyoxyethylene stearates, a saturated polyglycolyzed glyceride andcombinations thereof.
 13. A composition consisting of an isolatedfraction of mastic gum and a pharmaceutically acceptable carrier,wherein the fraction is characterized in that it is soluble in at leastone polar organic solvent and in at least one non-polar organic solvent,and wherein said fraction is substantially devoid of compounds which aresoluble in said polar organic solvent but insoluble in said non-polarorganic solvent, wherein said polar organic solvent is an alcohol,wherein the carrier is an oil selected from the group consisting ofcottonseed oil, almond oil, canola oil, coconut oil, corn oil, grapeseed oil, olive oil peanut oil, saffron oil, sesame oil, soybean oil andcombinations thereof.
 14. The composition of claim 13, wherein thealcohol is selected from the group consisting of ethanol, methanol,propanol, isopropanol, 1-butanol, 2-butanol, sec-butanol, t-butanol,1-pentanol, 2-pentanol, 3-pentanol, neopentanol, 3-methyl-1-butanol,2-methyl-1-butanol, 3-methyl-2-butanol, 2-methyl-2-butanol.
 15. Thecomposition of claim 13, wherein the at least one non-polar organicsolvent is selected from the group consisting of acyclic or cyclic,saturated or unsaturated aliphatic hydrocarbons and aromatichydrocarbons, each of which is optionally substituted by one or morehalogens, and combinations thereof; or wherein the non-polar organicsolvent is selected from the group consisting of C5-C10 alkanes, C5-C10cycloalkanes, C6-C14 aromatic hydrocarbons and C7-C14 perfluoroalkanes,and combinations thereof; or wherein the non-polar organic solvent isselected from the group consisting of pentanes, hexanes, heptanes,octanes, nonanes, decanes, cyclopentane, cyclohexane, cycloheptane,benzene, toluene, xylene, and isomers and mixtures thereof; or whereinthe non-polar organic solvent is hexane.
 16. The composition accordingto claim 13, wherein the isolated fraction is obtained by a processcomprising the steps of: (a) contacting plant material with at least onealcohol, wherein the plant material is selected from the groupconsisting of resin, leaves, twigs, roots, flowers, seeds, buds, bark,nuts, roots and combinations thereof; (b) isolating a fraction whichremains soluble in the at least one alcohol solvent (c) optionallyremoving said alcohol solvent; (d) treating the soluble fractionobtained in step (b) or (c) with at least one non-polar organic solvent;(e) isolating a fraction soluble in said nonpolar organic solvent; and(f) optionally removing said nonpolar organic solvent; wherein steps (a)to (c) and steps (d) to (f) are each independently optionally carriedout for a number of cycles; so as to obtain an isolated fraction ofmastic gum.
 17. The isolated fraction according to claim 16, whereinsaid process further comprises the step of size fractionating thefraction obtained in step (c) or step (f); or wherein either or both ofsteps (c) and (f) comprise removing the solvent by a means selected fromthe group consisting of rotary evaporation, application of high vacuumand a combination thereof.
 18. The composition according to claim 13,wherein the fraction is substantially devoid of terpene compounds whichare soluble in said polar organic solvent and insoluble in saidnon-polar organic solvent.
 19. The composition according to claim 13,wherein the fraction contains polymeric myrcene having a number averagemolecular weight in the range of at least about 1,000 to about 20,000.20. The composition according to claim 13, wherein the isolated fractionof mastic gum is from about 0.01 to about 25% (w/w) of the total weightof the composition.
 21. The composition according to claim 13, which isin a form suitable for cosmetic or dermatologic administration; or in aform suitable for administration by a route selected from the groupconsisting of oral, topical, parenteral, intramuscular, subcutaneous,intradermal, vaginal, rectal, intracranial, intranasal, intraocular,auricular, pulmonary intralesional, intraperitoneal, intraarterial,intracerebral, intracerebroventricular, intraosseus and intrathecal; orin a form selected from the group consisting of a capsule, a tablet, asuppository, a suspension, an ointment, a cream, a lotion, a solution,an emulsion, a film, a cement, a powder, a glue, an aerosol and a spray.